JP4577666B2 - Sex-specific fertilization of mammals with a low number of sperm cells - Google Patents

Abstract

Artificial Insemination is achieved for sexed mammalian offspring in a commercially practical manner and with dosages of insemination sperm which were not previously thought to be practical for commercial implementation. Improved insemination systems particularly adapted to use for sex-selected sperm sorting include systems which achieve superovulation and then multiple embryo production with sexed embryos. These systems combine with other techniques, including techniques for enhanced sheath fluid (3) and other strategies which minimize stress on the sperm cells (18), and potentially, a 2.9 percent sodium citrate sheath solution for bovine species and a hepes bovine gamete media for equine species. Improved collection systems (14) and techniques for the process are describeb so that commercial application of sperm samples as well as the resulting animals may now be achieved in the field.

Description

本発明において相互作用し得る１つの他の局面は、含まれる細胞が、異常な感受性を経験し得るという事実である。１つの点に関して、これは精子細胞が非修復細胞である細胞のクラス内であるという事実に起因し得る。すなわち、それらは、それ自体を修復する能力を有さず、それ故、それらは、フローサイトメーターまたは他の取扱い設備に対して代表的であるよりも感受的に処置されることを必要とし得る。従って、フローサイトメーター、または他の分離デバイスが精子細胞の供給源を確立するように作用する場合、この増強は、特に適用可能であることが適切であり得る。精子細胞のような細胞のクラスに独特であり得る、別の可能性のある関連する局面は、それらのＤＮＡが非修復、非複製、および非転写であるという事実である。これらの因子のいずれかは、効果を示し得、そしてそれらは、個々または一緒のいずれかで関連し得る。従って、本発明の教示は、非修復、非翻訳、非転写細胞である、全ての配偶子細胞またはウイルスなどにさえも適用し得る。
【０２１３】
また重要であり得るフローサイトメーター処理の別々の局面は、選別された後に化学的および物理的の両方で細胞を適切に処理する事実である。図４に示されるように、ドロップ（８）内の細胞をコレクター（１４）内に獲得するために、コレクターを構成する容器は、適切なサイズであり、その結果細胞と容器自体との間の衝撃を回避するいくつかの手段として作用することが重要であり得る。細胞が容器の底部にぶつからないようにするため、細胞を収集する容器の底部に最初のコレクター流体（１７）を置くことは公知であるが、容器内への細胞の衝突に起因する流動の提示ならびに回避不能なはね（ｓｐｌａｓｈｉｎｇ）において変動に処理を施すための容器の単純な広さは、この結果を増強するために使用され得ることが明らかである。１つの点に関して、これは緩衝要素として作用し得、その結果機械的に繊細であり得る細胞、すなわち、衝撃により破壊するかまたは損傷を受け得る細胞は、適切に処理され得る。従って、サイトメーター供給源が、選別されるべき細胞のように物理的に繊細な細胞である細胞を確立する場合、開口部の広さ（１８）が細胞との接触を回避する様式において容器の壁を配置するために供する、広い収集管のようないくつかの型の緩衝要素を提供することは重要であり得る。従って、この管は、側壁にそれほど近接して存在しないので、選別されるそれらの細胞と管壁との間に接触の任意の有意な見込みが存在する。この様式において、コレクター流体（１７）に加えて、同様に広い収集管を備えることが所望される。おそらく、コレクター（１４）の部分として供する容器に広い開口部を単に提供することは、有意であり得る。精子細胞の高速分類を利用する適用のために、少なくとも１５ミリメーターの内部直径の開口部を有する容器を提供することが有意であると考えられることが見出されている。具体的に、このような適用において１４ｍｌのＦａｌｃｏｎ試験管を利用する場合、コレクター（１４）の結果として細胞への最小の物理的損傷が発見されている。
【０２１４】
１４ｍｌのファルコン試験管ですらも最適ではないかもしれないことに注意すべきである。特に、流れの幾何学に適した収集容器（すなわち、「流れ適合容器（ｓｔｒｅａｍ−ｍａｔｃｈｅｄ ｃｏｎｔａｉｎｅｒ）」）を設計することが最良であり得ると考えられる。この流れ適合容器は、以下の特徴のいずれかまたは全てを有し得る：相対的に広い開口部、楕円形に成形された開口部、現在関わっているものより小さな高さ：幅の比、角度がつけられているか、またはそうでなければ落下流に対して平行な側壁を示し得るように調整された体裁など。ボールベアリングなどのような可動要素または媒体のような取り付け要素を提供して、試験管の可変性の配向を、収集することが所望される落下流に適合させることもまた望ましい。さらに、既存の試験管（「ファルコン型」試験管として記載される）のような容器の分類についての物理的特徴としては、試験管の幅だけでなく試験管が作製される材質（例えば、細胞が固着しないポリスチレン）もまた挙げられ得る。（これらの材質選択は、１４ｍｌファルコン試験管について周知である。）従って、この容器およびその収集液はまた、緩衝要素として作用して、細胞への物理的損傷を最小限にし得る。これは、サイズによっては、有意な希釈効果なく精子の適切な数の収集を容易にするためにもまた作用し得る。
【０２１５】
コレクター液（１７）の別の局面は、細胞に対する化学的ストレスを最小にするためにもまた作用し得るという事実であり得る。１つの点では、選別の前後両方で細胞に栄養素を与えることが重要であり得るので、コレクター液（１７）は、調整されたレベルの栄養素を提供するように選択され得るため、この栄養素レベルは選別の前後両方で平衡化される。２％卵黄レベルで卵黄クエン酸塩の栄養素が利用されるウシ精子に関しては、６％卵黄クエン酸塩レベル（すなわち、クエン酸塩溶液中に６％の卵黄含量）を利用することが良好な結果を提供することが発見された。これは、選別事象の前後に存在する容量の結果である。コレクター液（１７）は、約２ｍｌの容量で（選別前に）開始され得る。選別事象によって、この容量の約２倍が添加され得（最初の開始容量の３倍で終了する）、（目詰まりおよび他のフローサイトメーターの考慮すべき事項に起因して）溶液中に卵黄クエン酸塩はほとんどない。従って、存在する卵黄クエン酸塩の量のレベルという点では、最終結果は、開始結果、すなわち、含まれる容量に起因して、クエン酸溶液中に２％卵黄クエン酸含量に等しくあり得る。従って、コレクター液（１７）は、最終コレクター液環境を作り出すように選択され得、この最終コレクター液環境は、最初の栄養素または他の流体環境と平衡化される。この様式で、コレクター液は、細胞が供される時間および組成のレベルの変化を最小にするために作用し得る。もちろん、これらの流体環境は、フローサイトメーター内で示されていてもよいし、ある他の重要な時間に存在していてもよく、重要な点は、細胞の寿命における任意の時点で細胞が供されるストレスをほとんど最小化しないことである。さらに、最初の化学物質量が変動され得る（例えば、クエン酸塩中のパーセント卵黄含量は、上下に変動され得る）ので、同様に、開始収集液環境または種々の容量もまた、最終結果が同じであるように変動され得る。従って、選別プロセスを開始する前に、コレクター液は、クエン酸塩溶液中６％卵黄含量で存在し、そして選別事象が終了した後には、コレクター液（性特定精子を有する）は、結果的に最初の栄養素含量と同様にクエン酸塩溶液中２％卵黄含量になり得る。
【０２１６】
後者の使用では、これらの精子細胞は、他の理由のためにクエン酸塩液中２０％卵黄含量に対して処理され得る。しかし、これらの変更は、全体的な受精プロセスの公知の部分に過ぎないように、細胞に対するストレスを提供するとは認められない。もちろん、このレベルは当業者によって容易に理解され得るので、変動され得るが、２０％卵黄クエン酸塩緩衝液は、以下のように構成され得る：
Ｉ．最終組成：
８０％ クエン酸ナトリウム溶液（７２ｍＭ）
２０％ （容量／容量）卵黄
ＩＩ．１Ｌ用の調製：
Ａ．クエン酸ナトリウム溶液
１．１，０００ｍｌ容積フラスコ中に２９．０ｇのクエン酸ナトリウム二水和物（Ｎａ₃Ｃ₆Ｈ₅Ｏ₇・２Ｈ₂Ｏ）を入れる
２．最終容量が１，０００ｍｌになるように脱イオン水またはナノピュア（Ｎａｎｏｐｕｒｅ）水を添加する
３．瓶に移し、１５ｌｂｓ圧力（２４５Ｆ°）で少なくとも３０分間オートクレーブにかける
ａ．蒸発を最小にするための条件を使用して溶液をオートクレーブにかける（緩くふたをする）
ｂ．水が沸騰して外に漏れないように注意する
４．室温でゆっくり冷却する
５．密栓して５℃の低温室で保存する
Ｂ．卵調製物
１．新鮮な鶏卵を優良な商業供給源から得る
２．埃がないように卵を洗浄し（過剰な界面活性剤は使用しない）、そしてすすぐ
３．卵を７０％エタノールに２〜５分間浸漬する
４．卵を取り出し、乾燥させ（または拭いて乾燥させ）、そして清潔なタオル上で保存する
Ｃ．エキステンダーの調製
１．清潔な滅菌ガラス製品を使用する
２．Ａ−画分（グリセロールなし画分）
ａ．１，０００ｍｌのメスシリンダーに８００ｍｌの２．９％クエン酸ナトリウム溶液を入れる
ｂ．エキステンダーのグリセロールなし画分（Ａ画分）についての抗生物質レベルは、以下のとおりであり得る；
ｉ．タイロシン＝１００μｇ／ｍｌ
ｉｉ．ゲンタマイシン＝５００μｇ／ｍｌ
ｉｉｉ．リンコ−スペクチン＝３００／６００μｇ／ｍｌ
ｃ．２００ｍｌの新鮮卵黄を以下に概説するように添加する（Ｄ節）
ｉ．徹底的に混合する
ｄ．これは、２０％卵黄およびウシ精子に対して非毒性であることが公知の濃度の抗生物質を有する２．９％クエン酸ナトリウムに基づいたＡ画分エキステンダーを提供する
ｅ．エキステンダーは、一晩５℃で保存され得る。
【０２１７】
ｆ．次の日に上清（上部の８００ｍｌ）をデカントする
ｇ．次の日の使用前に３７℃に暖める
Ｄ．緩衝化溶液に卵黄を添加するために、以下の手順を十分に行う
１．卵を十分に洗浄し、清潔にする（上記Ｂを参照のこと）
２．卵を割り、卵黄分離器を用いて卵白と卵黄を分離する。あるいは、卵黄を卵の殻に左右交互に２〜３回出し入れする。卵黄膜を破らないこと
３．卵黄を１５ｃｍの滅菌濾紙上におく
４．緩衝液を入れたメスシリンダーの上に濾紙を配置し、（卵黄膜を破りながら）卵黄を絞り出し、金で処理した（ｇｏｌｄｅｄ）濾紙からメスシリンダーへ流出させる。代表的には、約１２〜１５ｍｌの卵黄が１つの卵から得られ得る。
【０２１８】
本発明の種々の因子で相互作用し合い得る別の局面は、人工受精などのために低用量の精子を利用する局面である。雌雄識別した、人工受精の局面のさらなる背景は、「Ｐｒｏｓｐｅｃｔｓ ｆｏｒ Ｓｏｒｔｉｎｇ ＭａｍｍａｌｉａｎＳｐｅｒｍ」、Ｒｕｐｅｒｔ Ｐ．ＡｍｍａｎおよびＧｅｏｒｇｅ Ｅ．Ｓｅｉｄｅｌ，Ｊｒ．，Ｃｏｌｏｒａｄｏ Ａｓｓｏｃｉａｔｅｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ（１９８２）（本明細書中に参考として援用される）に見いだされ得る。言及したように、自然な受精は、何十億ものオーダーの精子数が関与する。代表的な人工受精は、現在では、ウシ種に関しては数百万の精子で行われ、そしてウマ種に関しては数億の精子で行われる。用語「低用量」によって、受精事象で用いられる精子の投与量が、代表的な人工受精事象で提供される代表的精子数の１／２より少ないか、または好ましくはそれの約１０％より少なくさえあることを意味する。従って、用語「低用量」は、代表的な人工受精投与量の状況で考えられることであり、絶対数としてもまた考えられる。現在百万〜一千万の精子が提供されるウシ精子に関しては、低用量プロセスは、約５００，０００精子数の絶対数、またはおそらく３００，０００精子以下と考えられ得る。事実、本発明の技術を利用することによって、良好な割合での人工受精の成功は、４８Ｔｈｅｒｉｏｇｅｎｏｌｏｇｙ １２５５（１９９７）で公開された論文「Ｕｔｅｒｉｎｅ Ｈｏｒｎ Ｉｎｓｅｍｉｎａｔｉｏｎ ｏｆ ＨｅｉｆｅｒｓＷｉｔｈ Ｖｅｒｙ Ｌｏｗ Ｎｕｍｂｅｒｓ ｏｆ Ｎｏｎ−ｆｒｏｚｅｎａｎｄ Ｓｅｘｅｄ Ｓｐｅｒｍａｔｏｚｏａ」（これは、本明細書中に参考として援用される）に示されるように、１００，０００および２５０，０００精子（それぞれ妊娠率は４１％および５０％）の受精のレベルで示された。精子細胞は、希釈に対する感受性を示すようであるため、これらの結果は、本発明の種々の技術に関して、低用量の精子サンプルの利用に対して特定の相互依存性を示し得る。絶対数は、種依存性であり得る。ウマ種では、たとえ、約二千五百万、一千万、五百万、または百万の精子より少ないにしても低用量プロセスが考えられ得る。
【０２１９】
重要であり得る別の局面は、本発明の（または他の）技術によって精子の雌雄識別が人工受精システムで利用されるという事実である。従って、フローサイトメーター技術に関しては、コレクター（１４）を使用して、人工受精のための精子を提供するときに、本発明の技術は、特に関連し得る。さらに、人工受精の使用および低用量環境での使用の両方の組み合わせは、ともに相乗効果を奏し得、これは、本発明の種々の技術を特に適切にすることが可能である。当然、雌雄識別された精子は、ただ人工受精態様だけではなく、インビトロ受精技術などのような他の技術においても利用され得る。
【０２２０】
フローサイトメトリーの使用または他の分離技術によって動物を収集、選別、および実際に人工受精するプロセスは、種々の工程を包含する。ウシの人工受精の状況で、第１に、精液を人工膣を使用してウシから収集する。これは、約１５億精子／ｍｌの割合である。この生の精液を分光光度計を使用してチェックして、濃度を評価し得る。そしてこの精液を検鏡によって評価して、適切な運動性および生存性基準を満たすことを確実にし得る。次いで、抗生物質が添加され得る。結果として、最初のサンプルは、１回の射精あたり約６０〜７０％の前進運動性を有し得る。処理工程に関しては、ある型のＴＡＬＰ（タイロードアルブミンラクテートピルベート）による希釈を用いて、約１億／ｍｌの管理可能なレベルで（フロー分析について）精子数を入手し得る。ＴＡＬＰは、精子細胞に栄養を与えるだけでなく、精子を染色工程のために機能亢進させ得る。染色前に、ウマ種のようないくつかの種では、遠心分離が行われ得る。染色工程を、多重染色または単色染色プロトコルに従って行い得、後者は、Ｊｏｈｎｓｏｎ特許および関連技術である。染色工程を、適切な栄養素環境を作り出すためにエキステンダーもまた調節しながら行い得る。ウシの適用において、これは、染色後すぐにクエン酸塩溶液中に約２０％卵黄含量を添加する工程を包含する。さらに、精子細胞を染色することにおいて、ある範囲まで予測され得るよりも高い染色量によって、より良好な結果が達成されることが発見された。これは、高濃度染色工程が、数１０μＭ含量の染料量（例えば、Ｈｏｅｃｈｓｔ ３３３４２染料が３８μＭ含量で使用される以下の実施例で議論される）を使用する工程を包含し得る。
【０２２１】
染料を添加した後のインキュベーション期間としては、例えば、約一億精子細胞／ｍｌの濃度で染料取り込みを促進するために３４℃で１時間インキュベートする工程を用い得る。次いで、それらを濾過して、精子細胞の凝集を取り除き得る。次いで、約一億精子細胞／ｍｌの所望の選別濃度への希釈または拡大が行われ得る。次いで、先に議論された種々の技術に従う選別工程が行われ、ここから精子細胞が収集相で回収され得る。先に言及されたように、収集は、約２％卵黄クエン酸塩濃度含量（ウシ種について）を有するサンプルで生じ得る。次いで、このサンプルを、シース液および保存液が除去され得る後に遠心分離によって、約３００〜５００万精子細胞／ｍｌに濃縮し得る。次いで、最終的な拡大は、２０％卵黄クエン酸塩またはＣｏｒｎｅｌｌ Ｕｎｉｖｅｒｓａｌ Ｅｘｔｅｎｄｅｒなどのいずれかで達成され得る。Ｃｏｒｎｅｌｌ Ｕｎｉｖｅｒｓａｌ Ｅｘｔｅｎｄｅｒは、１０００ｍｌについて以下の組成を有し得る：
１４．５ｇ クエン酸ナトリウム二水和物
２．１ｇ ＮａＨＣＯ₃
０．４ｇ ＫＣｌ
３．０ｇ グルコース
９．３７ｇ グリシン
０．８７ｇ クエン酸
２０％卵黄組成については、上記調製物の８００ｍｌおよび約２００ｍｌの卵黄を使用し得る。
【０２２２】
この最後の拡大後に、３００〜５００万精子／ｍｌ（ウシ種について）が生じ得る。次いで、このサンプルを精子の代謝を緩慢にするように、そしてより長い期間にわたる使用を可能にするように冷却され得る。次いで、ウマ種では、このサンプルは、当業者がよく理解するように、卵管プロセスまたは他の受精プロセスで使用され得る。ウシの精子では、サンプルは、所望の投与レベルまでさらに１回希釈され得る。希釈は、精子細胞の生存性に影響をもたらし得るために、より少量のサンプルを提供することによって希釈のレベルがあまりに大きくなり過ぎることを避けることが適切であり得ることが見いだされた。それにもかかわらず、使用されている分離技術のうち、現在では、約３００，０００精子／０．１８４ｍｌの低投与量が達成され得る。さらに、約５％のレベルで精漿のレベルを維持することが所望され得るが、この必要性の結果は、現在では結びつけられている。次いで、精子細胞試料は、人工受精に使用するためのストローに配置され、そして受精させるために経産牛または未経産牛に輸送され得る。
【０２２３】
都合のよいタイミングでの人工受精を達成するために、未経産牛または経産牛の発情期を、当該分野で周知の技術に従って、プロスタグランジンＦ２_αの利用のような公知の技術により同期化させ得る。この後者の内容は、論文「Ｐｒｏｓｔａｇｌａｎｄｉｎ Ｆ２_α−Ａ Ｆｅｒｔｉｌｉｔｙ Ｄｒｕｇ ｉｎ Ｄａｉｒｙ Ｃａｔｔｌｅ？」１８ Ｔｈｅｒｉｏｇｅｎｏｌｏｇｙ ２４５（１９８２）（これは、本明細書中に参考として援用される）で議論されるように未経産牛での受精能の増強を潜在的に達成することが報告されたという点で特に価値があり得る。最近の結果では、前述の事項は維持されなかったが、本発明は、雌雄識別された低用量の受精の状況で特別の生存性を実証することであり得る。次いで、ウシ種に関しては、人工受精は、子宮角内深くに精子細胞を配置する胎芽移植機器の使用によって達成され得る。これは、人工受精において代表的に使用されるピーク時ではなく、むしろピーク時の１２時間後のようないくらか遅い時期に達成され得る。なぜなら、雌雄識別された人工受精に関する受精は、わずかにより遅く生じ得るいくらかの可能性があるからである。胎芽移植機器の利用は、このような低用量の雌雄識別された受精に関して、子宮壁が非常に敏感であり得るために使用され得る。
【０２２４】
さらに、この技術を組み合わせて、さらにより効率的な生産を達成し得る。特に、高速選別および雌雄識別された胚の低用量人工受精を可能にする、たった今発明されたプロセスの各々は、過排卵動物において実行可能である。過排卵は、排卵誘発剤（ｓｕｐｅｒｏｖｕｌａｔｏｒｙ ｐｈａｒｍａｃｅｕｔｉｃａｌ）の使用または他の任意の技術によって達成され得る。排卵誘発剤は、例えば、正常よりも多く卵の生成を達成するために一連の反応によって直接または間接に作用し得る。過排卵との組み合わせは、以前、過排卵がこのような組み合わせを妨げると認められたので驚くべきことである。精子輸送は、過排卵処理されたウシで損なわれるので、動物を複数の機会でおよび／または複数の用量の精液で、頻繁に人工受精していた。また、精液を雌雄識別する以前の手順は、比較的緩慢であった；従って、これらの新たな技術の組み合わせを用いて、過排卵誘発剤（例えば、ＦＳＨ（卵胞刺激ホルモン））処置ウシの、合計６００，０００の雌雄識別した未凍結精子を用いた１回の人工受精後の受精率を決定することは、興味深いことである。
【０２２５】
例示として、１２頭のＡｎｇｕｓ交雑未経産牛を、標準的手順を使用して過排卵処理した：６、６、４、４、２、２、２、および２ｍｇのＦＳＨを発情周期の９日目と１２日目の間で始まる半日間隔で筋肉内注射した；２５ｍｇおよび１２．５ｍｇのプロスタグランジンＦ−２αを６回目と７回目のＦＳＨ注射とともに筋肉内注射した。未知の受精能のウシからの精子をＨｏｅｃｈｓｔ ３３３４２で染色し、次いで、ＭｏＦｌｏ（登録商標）フローサイトメーター／セルソーターを使用して選別し、７００〜８００の生きた精子を各々の性／秒で得た。平均選別純度は、所望される性の８９％であった。選別された精子を、５℃に冷却して６５０ｇ、１０分間で遠心分離することによって３．３６×１０⁶精子／ｍｌに濃縮し、そして４時間保存した。次いで、１８４μｌを０．２５ｍｌのプラスチックストローに入れ；この用量の半分を自動側壁開口胎芽移植シース（ａｕｔｏｍａｔｉｃ ｓｉｄｅ−ｏｐｅｎｉｎｇ ｅｍｂｒｙｏ ｔｒａｎｓｆｅｒ ｓｈｅａｔｈ）を使用して発情後２０〜２４時間後に各子宮角に人工受精した。胚を標準的な非外科的手順によって発情後７または１６日目に収集した。結果は、７日目と１６日目での収集との間およびＸおよびＹ選別精子で同様であった。胚を９頭の未経産牛から回収した。発生の正常段階で５２胚（平均４．３±５．３／ドナー）、１３の発達の遅れた胚および３１の未受精卵が存在した。４６胚が、Ｙ染色体特異的ＤＮＡ配列についてのプライマーを使用してＰＣＲによって雌雄識別され；そのうち４３（９３％）が意図された性であった。この研究は、少数の動物に関与しただけであるが、驚くべきことに、わずか合計６００，０００の（生存した）雌雄識別した未凍結精子での過排卵処置した未経産牛の人工受精は、従来の手順と同様の結果を与えた。上記のバリエーションはまた、フローサイトメーター手段以外によって選別する工程、他の様式で過排卵を達成する工程、他の様式で受精能を増加させる工程など（を含むが、これらに限定されない）で達成され得る。
【０２２６】
さらに、受精能を損なうことなく、ＤＮＡ含量に基づいて精子を雌雄識別する方法、高速フローサイトメーター／セルソーター、および合計５００，０００より少ない精子で未経産牛を人工受精する手順の調和が、わずか数年内にウシにおける生存可能な雌雄識別した精液産業の可能性を生じた。８５％より高い正確性で雌雄識別した精子についての多くの適用が存在する。おそらく、最も明白なのは、雌の群を置換するために、一方のサブセット（乳牛および肉牛の両方）のウシを人工受精すること、および逆のサブセット（乳牛および肉牛の両方）を肉用に生産された雄に対して全く異なるタイプのウシと交配させることである。上記の非常に重要なサブセットは、Ｘ染色体保有精子で未経産牛に人工受精して、雌ウシを生産することである。これは、主にサイズがより小さいことに起因して、雄ウシより難産の発生率は低くなる。さらに、若い乳用種牛を提供することは、未経産牛の優勢にはるかにより有効である。８５％より多い未経産牛を有することはまた、乳牛を管理することを可能にするために、それらは、平均すると、寿命あたり２頭の生存牛より少ない。これは、妊娠および分娩に関連する問題を減少させるためには魅力的なものである。肉生産の単一性別システム（ｓｉｎｇｌｅ ｓｅｘ ｓｙｓｔｅｍ）もまた可能になる。ここで、各雌をそれ自身が交換し、そして２〜３年の間で屠殺されるため、成長のためのシステム中ではるかにより高い栄養素を使用して、そして維持する割合はより低い。雌雄識別した精液は、インビトロ人工受精のために、および胎芽移植のために過剰排卵処理したウシを人工受精するために有用である。頻繁には、ある性のウシが他方よりかなり価値があり、そして胚を雌雄識別する正確な方法が利用可能であるにもかかわらず、それらは、時間がかかり、そして生成された胚のうちの半分は、あまり価値のない性である。正確に雌雄識別された精液は、雌雄識別の追加料金が低く、かつ受精能が最小限にしか損なわれない場合、ウシの人工受精に広範に適合すると推測される。人工受精される肉牛の割合は、おそらく、実質的に雌雄識別された精液での肉牛が増えるであろう。
【０２２７】
興味深いことに、このような受精が通常配置される子宮体内への受精よりも、子宮角内深くへの受精によって、より良好な結果が達成され得る。おそらく、このようにはるかに研究されたサンプルは、子宮角内深くで達成された場合に、同側性の受精と対側性の受精との間に違いを示さないこともまた驚くべきことである。深いことによって、その挿入は、胎芽移植機器を使用して、子宮角に良好に配置されることが理解されるべきである。同側性の受精と対側性の受精を使用して、結果が有意に異ならないようであるという事実は、本発明者らに、両方の受精の使用の提案を導き、その結果、適切な子宮角を同定するプロセスはもはや必要であり得ない。
【０２２８】
受精の結果として、当然のこととして、所望の性の動物が産生されることが所望される。この動物は、雌雄選別された精子標品の使用を介する先に議論した系に従って産生され得る。本発明の技術は、例えば、腹腔鏡検査的（ｌａｐｒｏｓｃｏｐｉｃ）受精、卵管受精などのような他の技術における適用を見い出し得ることがまた、理解されるべきである。
【０２２９】
実施例として、以下の実験が行われた。本明細書に記載される本発明のあらゆる局面のすべての使用ではなく、それらは、本発明の異なる局面を通して可能な実行の増強を示す。さらに、いくつかの実験の要約は、以前に引用した論文「非常に少数の非凍結および雌雄選別された精子を用いた未経産牛の子宮角受精」に含まれる。この論文は、本発明の有効性を示すデータのいくつかを要約する。実験については、雌雄選別された非凍結精子細胞を用いて行われ、以下のように高い成功を収めた。
【０２３０】
【実施例】
（実施例１）
Ａｎｇｕｓ未経産牛（１３〜１４月齢（ｍｏ）であり、そして中程度の体調）は、１２日間の間隔をあけて２５ｍｇのプロスタグランジンＦ−２αを用いて同期化させ、そして直立発情期（ｓｔａｎｄｉｎｇ ｅｓｔｒｕｓ）が観察された６〜２６時間後、受精させた。１４〜２６月齢の３頭の雄ウシから新鮮に収集した精液を、ＴＡＬＰ培地中で１時間、３４℃で、７５×１０⁶精子／ｍｌで、３８μＭ Ｈｏｅｃｈｓｔ ３３３４２中でインキュベートした。精子を、５０ｐｓｉで操作するＭｏＦｌｏ（登録商標）フローサイトメーター／セルソーターを使用して、そしてシース液として、２．９％ クエン酸Ｎａを使用して、１５０ｍＷでの３５１ｎｍおよび３６４ｎｍにおけるレーザー励起からの落射蛍光（ｅｐｉｆｌｕｏｒｅｓｃｅｎｃｅ）に基づいて、性染色体によって選別した。Ｘ染色体を有する精子（超音波処理した精子アリコートを回復させることによって確認されたように、約９０％の純度）を、約５００生精子／秒で、１００μｌ Ｃｏｒｎｅｌｌ Ｕｎｉｖｅｒｓａｌ Ｅｘｔｅｎｄｅｒ（ＣＵＥ）を含む２−ｍｌ Ｅｐｐｅｎｄｏｒｆ管に、２０％卵黄とともに収集した。収集した精子を、６００×ｇで１０分間遠心分離し、そして１．６３×１０⁶生精子／ｍｌでＣＵＥ中に再懸濁した。雌雄選別されていない精液のコントロールについて；Ｈｏｅｃｈｓｔ ３３３４２染色精子を、シース液で、９×１０⁵精子／ｍｌに希釈し、そして遠心分離し、そしてＣＵＥ中で１．６３×１０⁶の進行的に運動性の精子／ｍｌに再懸濁した。雌雄選別された精液および液体コントロール精液を５℃に７５分間冷却し、そして０．２５ｍｌストロー（１８４ｕｌ／ストロー）にロードした。ストローを、受精のために、分取の５〜９時間後に３〜５℃の温度制御飲料冷却機２４０ｋｍに移した。雌雄選別された精液および液体コントロール精液を、側開口青色シース（ｓｉｄｅ−ｏｐｅｎｉｎｇ ｂｌｕｅ ｓｈｅａｔｈｓ（ＩＭＶ））を使用して受精し、各々のストローの半分を、各々の子宮角に入れた（３×１０⁵生精子／未経産牛）。標準的なコントロールとして、同じ雄ウシ由来の精液を、標準的な手順によって０．５ｃｃストロー中で冷凍し（平均１５．６×１０⁶の運動性の精子／溶解後用量）、３５℃で３０秒間溶解し、そして子宮体中に受精させた。処置を、３頭の雄ウシおよび２人の受精者に対して、雌雄選別された精液および２つのコントロールについて３：２：２の受精の比で平均化した。妊娠を、胎仔もまた雌雄選別された場合（盲目的に）、受精の３１〜３４日後超音波で決定し、そして６４〜６７日後に確認した。データを表に示す。
【０２３１】
【表２】

^a,b異なる上付き文字を有する値の雌雄選別比は異なる（ｐ＜０．０２）。
【０２３２】
雌雄選別された精液を用いた妊娠率は、コントロールの８０％にすぎなかったが、この差異は統計学的に有意でなかった（＞０．１）。１回の妊娠は、雌雄選別されかつ凍結したコントロール群の各々において６４〜６７日で失われる；雌雄選別群中で１９胎児のうちの１８（９５％）は雌であり、そしてコントロール群の３０のうちの２０（６７％）は雌であった。液体精液コントロールは、５０倍より多い運動性の精液（全精子の１２０倍以上）を含む凍結精液コントロールに対して実質的に同一の妊娠率を産み出し、このことは、子宮角中への低用量受精の有効性を例証する。本発明者らは、フローサイトメーター技術および人工受精を使用して、ウシでの性比率を有意に変更した。
【０２３３】
同様に、雌雄選別されていない、非凍結の精子細胞を用いて１つの実験を行い、そして以下のように報告し得る。
【０２３４】
（実施例２）
実験の目的は、理想的な野外の条件下で、極度に少ない数の凍結精子を用いて未経産牛に受精させた場合に、妊娠率を決定することであった。上記の平均妊娠率である３頭のＨｏｌｓｔｅｉｎ雄ウシ由来の精液を、均質化ミルク、７％グリセロール（ＣＳＳ）エキステンダー、および５％相同精液漿中で、０．２５ｍｌフレンチストローあたり総精子２×１０⁵、５×１０⁵、または１０×１０⁶（コントロール）まで広げ、そして液体窒素蒸気に移して凍結させた。精液を、３７℃の水浴中で２０秒間融解した。１３〜１５月齢で３５０〜４５０ｋｇの体重のＨｏｌｓｔｅｉｎ未経産牛に、２５ｍｇのプロスタグランジンＦ−２−α（Ｌｕｔａｌｙｓｅ（登録商標））を、１２日間の間隔をあけて２回注入し、そして胎芽移植ストローガン（ｅｍｂｒｙｏ ｔｒａｎｓｆｅｒ ｓｔｒａｗ ｇｕｎ）および側開口シースを用いて受精させ、そして精液の半分は、発情期の検出の１２時間または２４時間後、各々の子宮角深くに入る。実験を５ヶ月にわたって５回の反復で行い、そして２人の受精者で平均された。繁殖の周囲温度はしばしば−１０℃から−２０℃であったので、受精器具を暖かく保つことに注意が払われた。妊娠を、発情から４０〜４４日後に、超音波を使用して、生存可能な胎仔の検出によって決定し、そして発情後５５〜６２日後に確認し；２０２の受胎産物のうちの４つが、これらの時期に失われた。５５〜６２日では、妊娠率は２×１０⁵、５×１０⁵、および１０×１０⁶総精子／受精（Ｐ＜．１）について、５５／１０３（５３％）、７１／１０１（７０％）および７２／１０２（７１％）であった。妊娠率は、雄ウシの間で異なっていたが（５９、６２、および７４％、Ｐ＜．０５）、実験者の間で（６４および６５％）、または発情後の受精回数（１２時間で６５％、２４時間で６４％、Ｎ＝１５３、各回）では異ならなかった。記載した方法を用いる、未経産牛における妊娠率は、受精あたり５×１０⁵および１０×１０⁶総精子を用いるのと同様であった。
【０２３５】
１つの実験がまた、雌雄選別された非凍結の精子細胞で行われ、そして以下のように報告され得る：
（実施例３）
精液を、Ａｔｌａｎｔｉｃ Ｂｒｅｅｄｅｒｓ Ｃｏｏｐｅｒａｔｉｖｅの雄ウシから収集し、ＨＥＰＥＳ緩衝化エキステンダー＋０．１％ＢＳＡで１：４に希釈し、そしてＭａｒｙｌａｎｄ、Ｂｅｌｔｓｖｉｌｌｅまで１６０ｋｍ（約２時間）輸送した。ここで精液を、周囲温度において、以前に記載された方法（Ｂｉｏｌ Ｒｅｐｒｏｄ ４１：１９９）を使用して、ＴＥＳＴ収量（２０％）エキステンダー中にフローサイトメトリーによって選別した。約９０％純度において、５〜６時間あたり各性の２×１０⁶精子までの選別速度を達成した。精子を、遠心分離（３００ｇで４分間）によって、２×１０⁶精子／ｍｌまで濃縮した。いくらかの精子を、相同な精液漿を含むエキステンダー中に選別した（最終濃度５％）。選別した精子を、飛行機でコロラドまで輸送し（約２，６００ｋｍ）、そして周囲温度または５℃のいずれかで保存した（Ｅｑｕｉｔａｉｎｅｒ（氷を含む仕切りを用いる遮蔽された装置）中で６時間以上輸送の間冷却した）。発情が検出されるよりも１１〜３６時間早くに、未経産牛または乳の出なくなったウシに、精子選別セッションの終わりの９〜２９時間以内に受精させた。精子（０．１ｍｌ中に１〜２×１０⁵）を、受精の際に超音波によって決定された最も大きい濾胞を有する卵巣に対して同側の子宮角に深く入れた。
【０２３６】
輸送され、周囲温度で保存された精子を受精させた場合に、１０頭の雌はいずれも妊娠しなかった。輸送の間に５℃で冷却された精子を受精させた２９頭の雌の内、１４頭が４週間、そして１２頭（４１％）が８週間の妊娠期間の間妊娠した。選別の最後の１０時間以内に受精させた２２頭の内１１頭が８週間妊娠したが、しかし選別の１７〜２４時間後に受精させた７頭の内１頭のみが妊娠した。精液漿の添加の有意な効果はなかった。１２の胎仔の内の１つは予測された性ではなく、１つは不明瞭であり、そして１０は、妊娠の６０〜７０日間の超音波検査によって決定されたように、予測された性であった。
【０２３７】
引き続いて、３３のさらなる未経産牛に、超音波検査の使用なしで各々の子宮角中に０．０５ｍｌ（上記のように広げた精液）を用いて受精させた；３例のみが受精後４週間妊娠し、そして１例のみが８週間妊娠したままであった。しかし、先の群からの異なる雄ウシを使用して、そしてすべての受精は、選別１８〜２９時間後に行った。別の雄ウシ由来の選別された精子を用いて、本発明者らの研究室から２００ｋｍの、さらなる３８例の未経産牛を同様に受精させた（選別約２２時間後）；これらのいずれも、受精後８週間妊娠しなかった。
【０２３８】
要約すると、フローサイトメトリーによって性染色体について選別された精子の人工受精によって、ウシにおいて妊娠を達成することは可能であり、そして胎仔の性比率は、選別された精子のＤＮＡ含有量についての再分析によって予測される性比率に近づく（９０％）。しかし、妊娠率は、長距離の精子の輸送を必要とした、これらの予備的実験において大きく変動した。受精率は、選別後１７時間で急激に減少したが、いくぶんかの混乱が存在した。なぜなら、異なった時間に異なった雄ウシを使用したからである。妊娠率に見られる変動は、雄ウシの違いによるものか、受精の技術によるものか、選別と受精の間の間隔によるものか、または他の因子によるものかを決定するために、さらなる研究が必要とされている。
【０２３９】
最後に、１つの実験がまた、雌雄選別されていない、非凍結精子細胞を用いて行われ、そして以下のように報告され得る。
【０２４０】
（実施例４）
この実施例の目的は、未経産牛に理想的な実験条件下で非常に少数の精子を用いて受精させた場合に、妊娠率を決定することであった。３頭のＨｏｌｓｔｅｉｎ雄ウシ由来の精液を、Ｃｏｒｎｅｌｌ Ｕｎｉｖｅｒｓａｌ Ｅｘｔｅｎｄｅｒおよび５％の均質な精液漿中で１×１０⁵または２．５×１０⁵精子／０．１ｍｌまで広げた；２．５×１０⁶全精子／０．２５ｍｌをコントロールとして使用した。十分に広げた精液を、０．１または０．２５ｍｌの受精用量を送達するために、改変した０．２５ｍｌプラスチックフレンチストロー中に詰めた。精液を５℃に冷却し、そして収集の２６〜５７時間後に使用した。Ｈｏｌｓｔｅｉｎ未経産牛１３〜１５月齢、体重３５０〜４５０ｋｇに、１２日間の間隔で、２５ｍｇ プロスタグランジンＦ−２α（Ｌｕｔａｌｙｓｅ（登録商標））を注射し、そして発情の検出から２４時間後に、胎芽移植ストローガンおよび側開口シースを用いて、１つの子宮角に受精させた。受精は、発情１２時間後超音波によって決定された最大の濾胞を有する側に対して同側性であった；排卵の側は、発情７〜９日後超音波によって黄体の検出によって確認した。妊娠は、発情４２〜４５日後超音波によって胎仔の検出によって決定した。この実験は、４回反復し、そして３人の受精技術者で平均した。排卵の側は、２２５未経産牛のうち２０５で正しく決定された（９１％）；驚くべきことに、妊娠率は、同側および対側の受精でほとんど同一であった。妊娠率は、１×１０⁵、２．５×１０⁵、および２．５×１０⁶精子／受精（Ｐ＞．１）について、３８／９３（４１％）、４５／８７（５２％）、および２５／４５（５６％）であった。技術者の間で、有意な妊娠率の違い（Ｐ＜．０５）が存在したが、雄ウシの間にはなかった。フローサイトメーターを用いて性によって選別された精子が、商業的な適用を有する記載した方法を用いて、受精あたりの精子の数を十分に減らすことが可能であり得る。
【０２４１】
上述のように、そして種々の実験から見られ得るように、野外は統計学的に根拠のあるものであり、従って種々のさらなる実験が、適切な組み合わせおよび制限のストラテジーを示すために行われ得る。従って、種々の影響の間の相乗作用（例えば、レーザー励起を用いる色素の効果および組み合わされた色素の効果が研究され得る例）が、さらに同定される。
【０２４２】
本出願に含まれる議論は、基本的な記載として働くことを意図している。読者は、特定の議論が明白にすべての可能な実施形態を記載しなくてもよいし；多くの代替手段が暗黙のものであることに気付くべきである。それはまた、本発明の一般的な性質について十分に説明しなくともよいし、そしてそれぞれの特色または要素が、実際により広い機能をいかにして示し得るか、または非常に多様な代替手段もしくは等価な要素をいかにして示し得るかを明白に示さなくともよい。かさねて、これらは、暗黙に本開示中に含まれる。本発明が、デバイスを指向する用語において記載されている場合には、各々のデバイスの要素は、暗黙に機能を行う。装置の請求項は、記載されるデバイスについて含まれてもよいだけではなく、また、方法またはプロセスの請求項が、本発明および各々の要素が行う機能を扱うために含まれ得る。記載または用語のいずれもが、提出され得る請求項の範囲を限定することを意図するものではない。種々の変更が、本発明の本質から離れることなくなされ得ることが理解されるべきである。このような変更はまた、明細書中に暗黙に含まれ得る。これらもなお、本発明の範囲にある。示される明白な実施形態、非常に多様な暗黙の代替的な実施形態、および広い方法もしくはプロセスなどの両方を含む広い開示が、本開示によって含まれる。
【０２４３】
さらに、本発明の種々の要素および請求項のそれぞれがまた、種々の様式において達成され得る。本開示は、このような各々のバリエーション（任意の器具の実施形態のバリエーションの実施形態、方法またはプロセスの実施形態、またはこれらの任意の要素のバリエーションにさえすぎない）を含むことが理解されるべきである。特に、本発明の要素に言及する開示として、各々の要素についての語が、たとえ機能または結果のみが同じであるとしても、等価な器具の用語または方法の用語によって表現され得ることが理解されるべきである。このような等価な、より広い、またはより一般的でさえある用語は、各々の要素または作用の記載に含まれるとみなされるべきである。このような用語は、本発明が権利を与えられる暗黙の広い適用範囲を明白にすることが所望される場合に置き換えられ得る。１つの例としてではなく、すべての作用が、その作用を獲得するための手段として、またはその作用を起こす要素として表現され得ることが理解されるべきである。同様に、開示された各々の物理的要素は、物理的要素が容易にする作用の開示を含むと理解されるべきである。この局面の１つの例としてではなく、「コレクター」の開示が、明白に議論されるか否かにかかわらず、「収集すること」の作用の開示を含むことが理解されるべきであり、そして逆に、「収集すること」の作用の開示のみが存在する場合、このような開示は、「コレクター」の開示を含むことが理解されるべきである。このような変更および代替的な用語は、本明細書中に明白に含まれることが理解される。さらに、最初に提示した請求項に加えて、その請求項は、少なくとも以下により広く取り組むために変更され得ることが理解されるべきである：ｉ）本明細書中に開示および記載されるデバイス、ｉｉ）開示および記載される関連方法、ｉｉｉ）そしてこれらのデバイスおよび方法の各々の、同様の、等価な、暗黙でさえのバリエーション、ｉｖ）開示および記載されるとして示される機能のそれぞれを達成するこれらの代替的な設計、ｖ）開示および記載されることを達成するために暗黙であるとして示される機能の各々を達成する、代替的な設計および方法、ｖｉ）別個のかつ独立した発明として示される各々の特徴、要素、および工程、ならびにｖｉｉ）上記の各々の種々の組み合わせおよび順列。
【０２４４】
本発明の理解を助けるために、種々の刊行された引用文献が役立ち得る。これらは以下に列挙され、そしてそれによって参考として援用される；しかし、記載がこの／これらの発明の特許化と矛盾しているとみなされ得る点で、これらの記載は明白に出願人によってなされたとはみなされない。強力に役立つ引用文献は、米国特許第５６６０９９７号；同第５５８９４５７号；同第５５１４５３７号；同第５４３９３６２号；同第５３４６９９０号；同第５１３５７５９号；同第５０２１２４４号；同第４９９９２８３号；同第４７４９４５８号；同第４６９８１４２号；同第４６８０２５８号；同第４５１１６６１号；同第４４４８７６７号；同第４３６２２４６号；同第４３３９４３４号；同第４２７６１３９号；同第４２２５４０５号；同第４１９１７４９号；同第４１５５８３１号；同第４０９２２２９号；同第４０８５２０５号；同第４０８３９５７号；同第４０６７９６５号；同第４００９２６０号；同第３８９４５２９号；同第３６８７８０６号；ＲＥ３２３５０号を含む。役立つ引用文献はまた、以下の刊行物を含み得る：
【０２４５】
【表３】

本明細書を通して、その文脈がそれ以外に必要としない限りは、語「ｃｏｍｐｒｉｓｅ」または、「ｃｏｍｐｒｉｓｅｓ」もしくは「ｃｏｍｐｒｉｓｉｎｇ」のようなバリエーションは、言及された要素もしくは整数、または要素もしくは整数の群を含むが、任意の他の要素もしくは整数、または要素もしくは整数の群を排除しないことを暗示することが理解される。
【０２４６】
【発明の効果】
本発明によれば、雌雄識別された受精が、より低い投与量で、現実的な商業環境の下で作動する様式において達成される。
【図面の簡単な説明】
【図１】図１は、本発明についてのフローサイトメーター分離技術によるソーターシステムの模式図である。
【図２】図２は、代表的なフローサイトメーターの自由落下領域において飛沫同伴した細胞の図である。
【図３】図３は、本発明の結果としてほぼ表れた差異を示す概念図である。
【図４】図４は、着陸帯領域において収集されるような選別した細胞流の図である。
【符号の説明】
３ シース液
１４ 収集システム
１８ 精子細胞[0001]
BACKGROUND OF THE INVENTION
The present invention relates generally to the field of sex selection of mammalian offspring. The present invention particularly relates to aspects of low dose artificial fertilization and increased egg production to produce the desired sex offspring. Specifically, the present invention provides a system for sorting sperm via flow cytometry for sex-specific and low-dose efforts in sorting techniques, techniques during artificial fertilization and resulting in increased ovulation, Regardless, it is related to achieving sexed artificial insemination at low doses.
[0002]
[Prior art]
It is desirable to select the sex of a particular offspring for an extended period of time. Beyond obvious psychological aspects, the actual sex selection of mammalian offspring is significant when considering its application to food-producing animals (eg cattle) as well as famous winner animals (eg horses) Have significant economic significance. This great hope has become a significant variety of efforts to achieve sex-selected offspring. Perhaps the effort that seemed most likely to achieve the desired result was the effort in sorting and selecting between X and Y sperm prior to fertilization.
[0003]
One challenge faced in efforts to sort X and Y sperm is the large number of sperm involved. In natural fertilization, sperm is produced in billions in some species; however, without artificial fertilization, a significantly larger number of sperm is still used. For example, artificial insemination techniques typically use 10 million to 5 billion sperm (depending on the species). Therefore, a significant number of sperm is necessary even in the environment of artificial insemination.
[0004]
Many methods have been attempted to achieve separation between sperm carrying the X chromosome and sperm carrying the Y chromosome. These methods vary from magnetic technology as disclosed in US Pat. No. 4,276,139 to columnar technology as disclosed in US Pat. No. 5,514,537, US Pat. No. 3,894,529, reissued patent 32350, Covers a range of gravimetric techniques as discussed in U.S. Pat. Nos. 4,092,229, 4,067,965, and 4,155,583. The electrical characteristics are also described in U.S. Pat. S. As shown in US Pat. No. 4,083,957, and combinations of electrical and gravimetric properties have been attempted as discussed in US Pat. Nos. 4,225,405, 4,698,142, and 4,749,458. Kinetic efforts have also been attempted as shown in U.S. Pat. Nos. 4,0092,060 and 4,339,434. U.S. Pat. Nos. 4,511,661 and 4,999,283 (including monoclonal antibodies), and U.S. Pat. Nos. 5,021,244, 5,346,990, 5,439,362, and 5,660,997 (including membrane proteins), and U.S. Pat. Chemical techniques such as those shown in US Pat. No. 3,687,803, US Pat. No. 4,191,749, US Pat. No. 4,448,767, and US Pat. No. 4,680,258 (including antibodies), and addition of serum components as shown in US Pat. No. 4,085,205. Although each of these techniques is shown as being highly efficient, in fact, none of these techniques currently produce the desired level of sex preselection. However, regardless of whether separation techniques are ultimately used, the naturally occurring sperm is numerous, and the competing combination of approaches that separate sperm carrying the X chromosome and sperm carrying the Y chromosome are: It would be desirable to develop the ability to achieve fertilization with fewer sperm.
[0005]
At present, there is only one quantitative technique that can be used to achieve separation of X- and Y-chromosome sperm, including individual fractionation and separation of sperm via flow cytometry techniques. This technique appeared to be possible as a result of advances and discoveries including differential dye absorption of X- and Y-chromosome sperm. This was first considered in US Pat. No. 4,362,246 and was significantly expanded through the technique disclosed by Lawrence Johnson in US Pat. No. 5,135,759. Johnson's technique of separating X- and Y-chromosome spermatozoa using flow cytometry was a very significant advance that allowed such commercial separation of sperm for the first time. Although still experimental, separation is significantly enhanced by the use of high speed flow cytometers (eg, MoFlo® flow cytometer manufactured by Cytomation, Inc.) and US Pat. No. 5,150,313. No. 5,602,039, No. 5,602,349, and No. 5,643,796, and various other patents, including International PCT Patent Publication No. WO 96/12171. Utilization of Cytomation's MoFlo® cytometer allows speeds to be greatly increased, while these speed increases are particularly appropriate considering the large number of sperm used often However, certain problems still remain. Despite nearly a 10-fold advance in the speed possible with MoFlo® flow cytometers, even shorter fractionation times are desired for several reasons. First, as a practical matter, it was discovered that sperm is a time critical cell. These lose their effectiveness the longer they remain unused. Second, the timing of collection, fractionation, and fertilization has made speed a commercially important item. Thus, the time-critical nature of sperm cells and processes has made speed an essential factor in achieving high potency and success rates.
[0006]
Other problems also exist, ranging from practical problems to virtual problems. In practical terms, it was desirable to achieve sex-specific sperm samples using inexpensive disposable components and materials. Also, in terms of cost, it was desirable to be able to achieve sorting (and collection and fertilization) with the result of the most efficient effort possible. Thus, for commercial production and success in this area, improvements that can only show increased efficiency may still be important. Related to the practical aspect of cost is the practical aspect of overall process sensitivity and sensitivity. In this regard, it is desirable to simplify this process and make the procedure as robust as possible so that operator error or skill can continue to be reduced. They also combined to make fertilization at lower doses even more desirable.
[0007]
In addition to the delicacy of this process, it has always been known that the sperm itself is a very delicate cell. Although this factor may appear to be easily understood at first glance, in fact the full degree of sensitivity of this cell has not yet been fully investigated. In general, in flow cytometry situations, most sorted cells or particles are often spherical or otherwise physically resistant to various abuses. This is not the case for sperm cells. Indeed, as disclosed by the present invention, processing via conventional flow cytometer technology may in fact not be acceptable for cytometric sorting of sperm cells in certain applications. Sensitivity is a matter of dilution, and the inherent need of a flow cytometer to individually isolate and distinguish each cell, as well as representative flow cytometry, Over pressure and other stresses applied to other materials. This may also indicate a unique factor of sperm cells. Because even though sperm cells seem to be able to pass through the flow cytometer and can be sorted without having macroscopically identifiable side effects, the cells themselves are actually less than optimal in the fertilization process. This is because it seems to be stressed in that it functions. Thus, factor interactions appeared to be related and resulted in unusual problems from the prospect of sperm cell sorting and ultimate use for artificial insemination.
[0008]
Another problem that remains (despite the great progress achieved by the Johnson patent and related technology) was that prior to the present invention, using sex-identified sperm, regardless of the separation technique used, The fact is that it has been extremely difficult to achieve lower dose fertilization. Historically, some low-dose fertilization has been achieved, but more likely in a theoretical or laboratory environment than in an environment where it appears to be or is applicable in commercial applications. there were. In this regard, the desire was not only to achieve low-dose fertilization, but rather to achieve low-dose fertilization with a pregnancy success rate comparable to the current high-dose artificial fertilization efforts that have not identified males and females. . Thus, the progress achieved by the inventors in artificial insemination that is both male and female and at low doses represents for the first time a significant advance that can enable commercial applications.
[0009]
Another problem faced by those skilled in the industry (in addition to the significant advances made by the Johnson patent and related technology) is that the problem itself, ie, artificial insemination at a high success rate, is a number of factors. Is one of the statistical properties that seem to interact. Thus, proposed solutions may include combinations of factors that have been shown to be necessary, either alone or in combination with other factors, after some thorough statistical study. Such a decision is further compromised by the fact that the results themselves vary from species to species, and due to the fact that testing and statistical sampling against a large enough database does not seem to meet initial efforts. It can be difficult to confirm. For these reasons, the present invention may also include a combination of factors that, either individually or in combination, may indicate a suitable solution for a given application. Accordingly, this disclosure is considered broad enough that various combinations and disseminations of the disclosed technology can be achieved. Unknown synergies may exist for other factors. Such factors can range from factors in the sorting or possibly flow cytometer process to factors in the collection and fertilization processes. Currently, research has been achieved primarily in bovine species, but it is not believed that these techniques are limited to such species, or such problems are limited to sperm cells only. The technique used may have application beyond just sperm cells to the area containing any sensitive member being screened, or simply minimizing the effects of flow cytometry stress when this member is screened. It seems.
[0010]
Interestingly, the present invention takes an approach to minimize sorting effects or stress on sperm cells, but others can increase pressure and demand for speed and other such performance by In fact, he seems to have acted away from this direction. In essence, the drivers for low-dose fertilization and rapid processing, in an individual or possibly interrelated manner, may present problems that are limited to each other. Thus, there is a need for fast and sex-identified low-dose fertilization that has long been felt but unsatisfactory, and the techniques and elements to perform have been available for a long time. In the past, progress or perhaps a combination of progress has been clearly overlooked by those skilled in the art. Perhaps to some extent, those skilled in the art can recognize that this problem involves the interaction of factors and the unique need for the type of cells involved in the field (sperm cells or possibly species-specific sperm cells). There wasn't. Interestingly, as the previous list of efforts in this discussion shows, substantial attempts have been made, but these clearly do not clearly understand the problems inherent in low-dose fertilization identified in male and female in these areas. And, since natural seeding events probably contain billions of sperm, it was hypothesized that there could be physical limitations on achieving artificial fertilization with numbers as small as four orders of magnitude. Thus, to some extent, it may not be surprising that there was an actual teaching away from the technical direction that we did. Perhaps this result can be further considered unexpected to some extent. This is because this result showed that low-dose artificial insemination with male and female discrimination could be achieved with a success rate comparable to that of high-dose artificial insemination without male and female discrimination. It can be even more surprising for some who actually combine the techniques and advances of the present invention to achieve the great results shown. Although each technique may be outlined in isolation as not surprising for some, in practice, slight changes, whether considered alone or in combination with other minor changes Seems to give significant progress to the final result.
[0011]
Thus, until the present invention, achieving success rates for male-female-identified low-dose artificial insemination is at the level of performance required, or in a simplified procedure that appears to be necessary to achieve commercial implementation. It was not possible. However, beyond the commercial level of male and female identified low-dose fertilization achieved to date, the present invention also allows for the achievement of improved performance and thus the desired end result, ie, on a commercial basis. A technique for facilitating male and female identified low-dose artificial insemination is disclosed.
[0012]
(III. Disclosure of the Invention)
Thus, the present invention claims the achievement of results as applied to pre-determine mammalian sex with low dose fertilization at a commercial level. The present invention also provides an improved sheath and collector system for sperm cell sorting to determine their sex through flow cytometer separation techniques. In this separation technique, sheath fluid as typically used in flow cytometers is replaced with a fluid that minimizes stress on the sperm cells as they are sorted. In addition, the collection system is improved to minimize both physical and chemical stresses experienced by sperm cells. Various techniques and materials are shown, but as those skilled in the art will readily appreciate, various combinations and substitutions are best suited for performance based on species, separation techniques, objectives, and other parameters involved in a particular processing application. Can be used in a manner that can be
[0013]
[Problems to be solved by the invention]
Accordingly, it is an object of the present invention to simply achieve sex-separated fertilization in a manner that operates at a lower dosage and in a realistic commercial environment. The goal is also to achieve better sorting for substances (eg sperm cells). A related objective is to minimize the impact the sorting function itself has on cells or other sensitive members that can be sorted. For flow cytometry sorting technology, the specific objective is to minimize the impact of sheath fluid on the cells and potential sheath fluid that acts positively to assist the cells in handling the various stresses involved. Is to provide. Similar objectives are particularly suitable for the separation of such sperm cells, generally into sperm cells, into bovine sperm cells, into equine sperm cells, and into X- and Y-chromosome-containing components. To provide a certain substance and technology. Similarly, one objective is to minimize the impact that the collection phase (eg, after sorting) has on the cells and to minimize physical and chemical impacts on such male and female identified sperm cells. It is to be. One aim is therefore to achieve a screening result with as little influence as possible.
[0014]
Another object of the present invention is to achieve low dose screened fertilization with a level and success rate comparable to the level and success rate of representative, male and female, high-dose artificial fertilization. Consistent with this goal, the goal is to present a general system for artificial insemination that can achieve this goal in a commercially practical manner. Thus, the prior goal of minimizing stress or potential damage to sperm cells is important. Sorting in a manner that provides both high speed and low stress sorting and is particularly adapted to sperm cell sorting in low dose situations is an important objective as well. It is also important to provide sheath fluids and other fluids that do not negatively affect sperm fertility and are compatible with artificial fertilization.
[0015]
Of course, further objects of the invention are disclosed throughout other areas of the specification and claims.
[0016]
[Means for Solving the Problems]
According to the present invention, a method of producing a mammal having a predetermined sex is provided, which method comprises the following steps:
a. Collecting sperm cells from males of mammalian species;
b. Determining the sexual characteristics of the plurality of sperm cells;
c. Sorting sperm cells according to determination of their sexual characteristics;
d. Establishing a fertilized sample having a small number of sperm cells compared to a representative artificial fertilization dose;
e. Inserting at least a portion of the fertilized sample into a female female of mammalian species;
f. Fertilizing at least one egg in a female of a mammalian species at a success level that is statistically comparable to a representative unsexed artificial fertilization dose; and
g. Producing a progeny mammal of the desired sex;
Is included.
[0017]
In one embodiment, the step of fertilizing at least one egg in a female of a mammalian species at a level of success that is statistically comparable to a representative artificial fertilization dose comprises Fertilizing at least one egg in the seed female at a success level selected from the group consisting of at least 35%, at least 41%, at least 50%, and at least 90%.
[0018]
In one embodiment, the step of collecting sperm cells from a male of a mammalian species comprises collecting sperm cells from a male of a mammalian species selected from the group consisting of cattle and horses.
[0019]
In one embodiment, the above steps of establishing a fertilized sample with a small number of sperm cells compared to a representative artificial insemination dose is provided in a representative unsexed artificial insemination event. Establishing a fertilized sample having no more than 10% of a representative number of sperm.
[0020]
In one embodiment, the above-described step of establishing a fertilized sample having a small number of sperm cells compared to a representative artificial fertilization dose comprises 100,000ofBovine fertilized sample of sperm cells, 250,000ofBovine fertilization sample of sperm cell, bovine fertilization sample of sperm cell of 300,000 or less, horse fertilization sample of sperm cell of 1 million or less, horse fertilization sample of sperm cell of 5 million or less, horse fertilization sample of sperm cell of 10 million or less And establishing a fertilization sample selected from the group consisting of horse fertilization samples of up to 25 million sperm cells.
[0021]
In one embodiment, the above-described step of inserting at least a portion of a fertilized sample into a female of a mammalian species, and at least one egg in a female of the mammalian species is a representative unsexed artificial The above steps of fertilizing at a success level that is statistically comparable to the fertilization dose are each accomplished in the field environment.
[0022]
In one embodiment, the step of inserting at least a portion of a fertilized sample into a female of a mammalian species in the field environment, and representative of at least one egg in the female of the mammalian species in the field environment. The above-described process of fertilizing at a statistically comparable success level for artificially fertilized doses that are not male and female identified is a rapid succession of a significant number of fertilized samples to a significant number of mammalian species females. And repeatedly inserting in farm or ranch conditions.
[0023]
In one embodiment, the mammal has a uterine horn and the step of inserting at least a portion of a fertilized sample into a female of a mammalian species comprises the fertilized sample both ipsilaterally and contralaterally. Inserting into the uterine horn of a female of mammalian species.
[0024]
In one embodiment, the mammal has at least one uterine horn and the step of inserting at least a portion of the fertilized sample into a female of the mammalian species inserts the fertilized sample deeply into the uterine horn. The process of carrying out is included.
[0025]
In one embodiment, the step of inserting at least a portion of the fertilized sample into a female female of mammalian species further comprises the step of inserting the fertilized sample deeply into the uterine horn.
[0026]
In one embodiment, the step of inserting at least a portion of the fertilized sample into a female female of mammalian species further comprises the step of inserting the fertilized sample into the uterine horn by use of an embryo transfer device.
[0027]
In one embodiment, the step of inserting at least a portion of the fertilized sample into a female female of mammalian species further comprises the step of inserting the fertilized sample into the uterine horn by use of an embryo transfer device.
[0028]
In one embodiment, the above-described step of inserting at least a portion of the fertilized sample into a mammalian seed female is a time during which the fertilized sample is generally considered optimal for a single artificial fertilization. Insertion step after 12 hours.
[0029]
In one embodiment, the step of establishing a fertilized sample having a small number of sperm cells as compared to a representative artificial insemination dose comprises establishing an unfrozen fertilized sample, wherein Wherein the step of sorting sperm cells according to the determination of their sexual characteristics is performed at the sorting time, and wherein the step of inserting at least a portion of the fertilized sample into the female female species is about from the sorting time. Do not delay more than 17 hours.
[0030]
In one embodiment, the step of establishing a fertilized sample having a small number of sperm cells as compared to a representative artificial insemination dose comprises establishing an unfrozen fertilized sample, wherein Wherein the step of sorting sperm cells according to determination of their sexual characteristics is performed at a sorting time, and the step of inserting at least a portion of a fertilized sample into a mammalian female species is about 10 hours from the sorting time. Do without delay.
[0031]
In one embodiment, the step of determining the sexual characteristics of a plurality of sperm cells comprises staining the cells with a high concentration of dye.
[0032]
In one embodiment, the step of determining the sexual characteristics of a plurality of sperm cells comprises staining the cells with a dye content of at least about 38 μM.
[0033]
In one embodiment, the above steps of determining sexual characteristics of a plurality of sperm cells, and the above steps of selecting sperm cells according to the determination of their sexual characteristics include the following steps:
a. Establishing a cell source supplying the cells to be sorted;
b. Chemically adjusting the sheath fluid to create a sheath fluid environment for the cells, wherein the sheath fluid is adjusted in both the pre-sort and post-sort cell fluid environments;
c. Sensing the characteristics of the cells;
d. Discriminating between cells having the desired sexual characteristics; and
e. Collecting cells having desired sexual characteristics;
Is included.
[0034]
In one embodiment, the above steps of determining sexual characteristics of a plurality of sperm cells, and the above steps of selecting sperm cells according to the determination of their sexual characteristics include the following steps:
a. Establishing a cell source supplying the cells to be sorted;
b. Chemically adjusting the sheath fluid to create a sheath fluid environment for the cells, wherein the sheath fluid is adjusted in both the pre-sort and post-sort cell fluid environments;
c. Sensing the characteristics of the cells;
d. Discriminating between cells having the desired sexual characteristics; and
e. Collecting cells having desired sexual characteristics;
Is included.
[0035]
In one embodiment, the above steps of determining sexual characteristics of a plurality of sperm cells, and the above steps of selecting sperm cells according to the determination of their sexual characteristics include the following steps:
a. Establishing a cell source supplying the bovine sperm cells to be sorted;
b. Establishing a sheath fluid comprising about 2.9% sodium citrate for bovine sperm cells;
c. Sensing the characteristics of the cells;
d. Discriminating between cells having the desired sexual characteristics; and
e. Collecting cells having desired sexual characteristics;
Is included.
[0036]
In one embodiment, the above steps of determining sexual characteristics of a plurality of sperm cells, and the above steps of selecting sperm cells according to the determination of their sexual characteristics include the following steps:
a. Establishing a cell source supplying the horse sperm cells to be sorted;
b. Establishing a sheath fluid containing Hepes buffered media for equine sperm cells;
c. Sensing the characteristics of the cells;
d. Discriminating between cells having the desired sexual characteristics; and
e. Collecting cells having desired sexual characteristics;
Is included.
[0037]
In one embodiment, the above steps of determining sexual characteristics of a plurality of sperm cells, and the above steps of selecting sperm cells according to the determination of their sexual characteristics include the following steps:
a. Establishing a cell source supplying the cells to be sorted;
b. Establishing a sheath fluid for the cells;
c. Sensing the characteristics of the cells;
d. Discriminating between cells having the desired sexual characteristics; and
e. Collecting cells having the desired sexual characteristics while buffering the cells from impact with the collection container;
Is included.
[0038]
In one embodiment, the above steps of determining sexual characteristics of a plurality of sperm cells, and the above steps of selecting sperm cells according to the determination of their sexual characteristics include the following steps:
a. Establishing a cell source supplying the bovine sperm cells to be sorted;
b. Establishing a sheath fluid for bovine sperm cells;
c. Sensing the characteristics of bovine sperm cells;
d. Discriminating between bovine sperm cells having the desired sexual characteristics; and
e. Collecting bovine sperm cells having the desired sexual characteristics in a citric acid collection solution containing about 6% egg yolk prior to initiating the collection step;
Is included.
[0039]
According to another aspect of the invention, a method of producing a mammal of the desired sex is provided, the method comprising producing the mammal using the process.
[0040]
In one embodiment, the method further comprises the step of rapidly sorting sperm cells.
[0041]
In one embodiment, the method further comprises the step of producing a plurality of eggs using an ovulation formulation, wherein at least one egg in the female of the mammalian species is represented by The above steps of fertilizing at a statistically comparable success level for artificially fertilized doses that are not male and female identified include fertilizing a plurality of eggs to produce a plurality of male and female identified embryos. .
[0042]
In one embodiment, the above step of producing a plurality of eggs using an ovulation formulation comprises injecting a dose of follicle stimulating hormone multiple times per day.
[0043]
In one embodiment, the step of injecting multiple doses of follicle stimulating hormone multiple times a day is approximately half a day increments between 6, 9, 4 and 4 between days 9 and 12 of the estrous cycle. Injecting follicle stimulating hormone comprising dosage levels of 4, 2, 2, 2, and 2 mg, and in the sixth and seventh doses of follicle stimulating hormone, 25 and 12.5 mg respectively. The method further includes injecting prostaglandin F-2-α.
[0044]
In one embodiment, the method comprises the following steps:
a. Staining male sperm cells;
b. Sorting by use of high-speed flow cytometry according to the sex of the sperm cells; and
c. A step of concentrating the sorted sperm cells;
In addition.
[0045]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, which includes the following:
  a. A cell source supplying cells to be analyzed by a flow cytometer;
  b. A sheath fluid source for creating a sheath fluid environment comprising about 2.9% sodium citrate for the cells;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that acts to sort cells having the desired characteristics; and
  g. A collector, in which cells having the desired characteristics are arranged,
Is provided.
[0046]
In one embodiment, the cell source comprises bovine sperm cells.
[0047]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, which includes the following:
  a. A cell source supplying cells to be analyzed by a flow cytometer;
  b. A sheath fluid source for creating a sheath fluid environment for cells containing a Hepes buffered medium;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that acts to sort cells having the desired characteristics; and
  g. A collector, in which cells having the desired characteristics are arranged,
Is provided.
[0048]
In one embodiment, the cell source comprises horse sperm cells.
[0049]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, which includes the following:
  a. A cell source supplying cells to be analyzed by a flow cytometer;
  b. A sheath fluid source that creates a sheath fluid environment for the cells;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that acts to sort cells having the desired characteristics; and
  g. A collector comprising a citrate collector fluid with cells having the desired characteristics disposed therein and comprising about 6% egg yolk;
Is provided.
[0050]
In one embodiment, the sheath fluid source comprises a solution comprising about 2.9% sodium citrate.
[0051]
In one embodiment, the cell source comprises bovine sperm cells.
[0052]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, which includes the following:
  a. A cell source supplying cells to be analyzed by a flow cytometer;
  b. A sheath fluid source that creates a sheath fluid environment for the cells;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that acts to sort cells having the desired characteristics; and
  g. A collector comprising a test tube having physical characteristics of a container in which cells having the desired characteristics are placed and adapted to flow;
Is provided.
[0053]
In one embodiment, the cell source comprises cells that are mechanically sensitive.
[0054]
In one embodiment, the cell source comprises cells that are over-responsive to the chemical composition of the surrounding fluid environment.
[0055]
In one embodiment, the collector is used to provide a low dose of sperm.
[0056]
In one embodiment, the nozzle, the oscillator, the cell sensing system, and the sorter identification system are part of a flow cytometer system, where the flow cytometer system comprises a high speed cell sorter.
[0057]
In one embodiment, the nozzle, the oscillator, the cell sensing system, and the sorter identification system are part of a flow cytometer system, where the flow cytometer system comprises a high speed cell sorter.
[0058]
According to another aspect of the present invention, a sex-separated sperm specimen is provided, which is produced according to the system.
[0059]
In one embodiment, the collector is used to provide a low dose of sperm.
[0060]
In one embodiment, the nozzle, the oscillator, the cell sensing system, and the sorter identification system are part of a flow cytometer system, where the flow cytometer system comprises a high speed cell sorter.
[0061]
In one embodiment, the collector is used to provide a low dose of sperm.
[0062]
According to another aspect of the present invention, there is provided a mammal produced using a sex-identified sperm specimen produced according to the above system.
[0063]
In one embodiment, the mammal is produced by the use of low dose sperm.
[0064]
In one embodiment, the nozzle, the oscillator, the cell sensing system, and the sorter identification system are part of a flow cytometer system, where the flow cytometer system comprises a high speed cell sorter.
[0065]
In one embodiment, the mammal is produced by the use of low dose sperm.
[0066]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, the system comprising:
  a. A cell source supplying the cells to be analyzed by a flow cytometer;
  b. A chemically tuned sheath fluid source that creates a sheath fluid environment for the cells that is selected to be conditioned in both the pre-sort and post-sort cell fluid environments;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that operates to sort cells having the desired characteristics; and
  g. A collector, in which cells having the desired characteristics are arranged,
Is provided.
[0067]
In one embodiment, the pre-sort and post-sort cell fluid environment comprises at least one hyperresponsive chemical composition to which the cells are particularly responsive, and the chemically conditioned sheath fluid supply The source minimizes changes to this overresponsive chemical composition.
[0068]
In one embodiment, the hyperresponsive chemical composition comprises a metabolic chemical composition.
[0069]
In one embodiment, the hyperresponsive chemical composition comprises citric acid.
[0070]
In one embodiment, the cell source creates the pre-sort cell fluid environment and the collector creates the post-sort cell fluid environment.
[0071]
In one embodiment, the cell source comprises non-repaired cells.
[0072]
In one embodiment, the cell source includes cells with non-transcribed DNA.
[0073]
In one embodiment, the cell source includes cells with non-replicating DNA.
[0074]
In one embodiment, the cell source comprises sperm cells.
[0075]
In one embodiment, the cell source comprises bovine sperm cells.
[0076]
In one embodiment, the cell source comprises horse sperm cells.
[0077]
In one embodiment, the cell source includes cells that are over-responsive to chemical compositions in a sheath fluid environment.
[0078]
In one embodiment, the collector is used to provide a low dose of sperm.
[0079]
In one embodiment, the low dose sperm comprises a dose of less than about 10% of a typical dose.
[0080]
In one embodiment, the sperm cells comprise bovine sperm cells, and the low dose sperm comprises a dose of less than about 500,000 sperm.
[0081]
In one embodiment, the sperm cells comprise bovine sperm cells, and the low dose sperm comprises a dose of less than about 300,000 sperm.
[0082]
In one embodiment, the sperm cells comprise horse sperm cells and the low dose sperm comprises a sperm dose of less than about 10 million.
[0083]
In one embodiment, the chemically conditioned sheath fluid source comprises a solution comprising about 2.9% sodium citrate.
[0084]
In one embodiment, the cell source comprises bovine sperm cells.
[0085]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, which includes the following:
  a. A cell source supplying cells to be analyzed by a flow cytometer;
  b. A sheath fluid source for creating a sheath fluid environment comprising a solution comprising about 2.9% sodium citrate for the cells;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that acts to sort cells having the desired characteristics; and
  g. A collector, in which cells having the desired characteristics are arranged,
Is provided.
[0086]
In one embodiment, the collector is used to provide sperm for artificial insemination.
[0087]
In one embodiment, the collector is used to provide a low dose of sperm for artificial insemination.
[0088]
In one embodiment, the chemically conditioned sheath fluid source comprises a solution comprising Hepes buffered media.
[0089]
In one embodiment, the cell source comprises horse sperm cells.
[0090]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, which includes the following:
  a. A cell source supplying cells to be analyzed by a flow cytometer;
  b. A sheath fluid source for creating a sheath fluid environment comprising a solution comprising hepes buffered media for the cells;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that acts to sort cells having the desired characteristics; and
  g. A collector, in which cells having the desired characteristics are arranged,
Is provided.
[0091]
In one embodiment, the collector is used to provide sperm for artificial insemination.
[0092]
In one embodiment, the collector is used to provide a low dose of sperm for artificial insemination.
[0093]
In one embodiment, the nozzle, the oscillator, the cell sensing system, and the sorter identification system are part of a flow cytometer system, where the flow cytometer system comprises a high speed cell sorter.
[0094]
In one embodiment, the high speed cell sorter sorts cells to be analyzed at a rate that sorts at least about 500 per second.
[0095]
In one embodiment, the high speed cell sorter is operated at a pressure of at least about 50 pounds per square inch.
[0096]
In one embodiment, the collector comprises a container that includes a buffer element.
[0097]
In one embodiment, the container comprises a wide collection tube.
[0098]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, which includes the following:
  a. A cell source supplying bovine sperm cells to be analyzed by a flow cytometer;
  b. A chemically tuned sheath fluid source that creates a sheath fluid environment comprising about 2.9% sodium citrate for the cells;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that acts to sort cells having the desired characteristics; and
  g. A collector, wherein cells having the desired characteristics are disposed therein, comprising a citrate collection fluid comprising about 6% egg yolk and used to provide a dose of less than about 500,000 sperm;
With
Here, these nozzles, oscillator, cell sensing system, and sorter identification system are part of a flow cytometer system, at a rate that sorts at least about 500 per second, Sperm cells to be analyzed are sorted and operated at a pressure of at least about 50 pounds per square inch.
[0099]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, which includes the following:
  a. A cell source supplying horse sperm cells to be analyzed by flow cytometer;
  b. A chemically tuned sheath fluid source that creates a sheath fluid environment for cells containing a Hepes buffered medium;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that acts to sort cells having the desired characteristics; and
  g. A collector, wherein cells having the desired characteristics are disposed therein, comprising a collection fluid comprising Hepes buffered media, and used to provide a sperm dose of less than about 10 million;
With
Here, these nozzles, oscillator, cell sensing system, and sorter identification system are part of a flow cytometer system, at a rate that sorts at least about 500 per second, Cells to be analyzed are sorted and operated at a pressure of at least about 50 pounds per square inch.
[0100]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, the system comprising:
  a. A cell source supplying the cells to be analyzed by a flow cytometer;
  b. Means for minimizing changes between the sheath fluid environment for the cells and the pre-sort and post-sort cell fluid environments;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that operates to sort cells having the desired characteristics; and
  g. A collector, in which cells having the desired characteristics are arranged,
Is provided.
[0101]
In one embodiment, the means for minimizing changes between the sheath fluid environment for the cells and the pre-sort and post-sort cell fluid environments comprises the sheath fluid.
[0102]
In one embodiment, the collector includes a collector fluid, and the means for minimizing changes between the sheath fluid environment for the cells and the pre-sort and post-sort cell fluid environments comprises: Contains this collector fluid.
[0103]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, which includes the following:
  a. A cell source supplying cells to be analyzed by a flow cytometer;
  b. A sheath fluid source that creates a sheath fluid environment for the cells;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that acts to sort cells having the desired characteristics; and
  g. A collector comprising cells having desired characteristics disposed therein and a chemically tuned collector fluid sheath fluid sharing source, wherein the collector fluid sheath fluid source is conditioned in a previous cell fluid environment. A collector, which is selected to create a collector fluid environment for the cells,
Is provided.
[0104]
In one embodiment, the collector fluid includes nutrients that are adjusted to average nutrient levels after completion of cell sorting.
[0105]
In one embodiment, the collector fluid comprises a citric acid solution containing about 6% egg yolk.
[0106]
In one embodiment, the cell source comprises cells that are overresponsive to the chemical composition in the collector fluid environment.
[0107]
In one embodiment, the cell source comprises sperm cells.
[0108]
In one embodiment, the cell source comprises bovine sperm cells.
[0109]
In one embodiment, the collector is used to provide a low dose of sperm.
[0110]
In one embodiment, the low dose sperm comprises a dose of less than about 10% of a typical dose.
[0111]
In one embodiment, the sperm cells comprise bovine sperm cells, and the low dose sperm comprises a dose of less than about 500,000 sperm.
[0112]
In one embodiment, the sperm cells comprise bovine sperm cells, and the low dose sperm comprises a dose of less than about 300,000 sperm.
[0113]
In one embodiment, the nozzle, the oscillator, the cell sensing system, and the sorter identification system are part of a flow cytometer system, and the flow cytometer system comprises a high speed cell sorter.
[0114]
In one embodiment, the high speed cell sorter sorts cells to be analyzed at a rate that sorts at least about 500 per second.
[0115]
In one embodiment, the high speed cell sorter is operated at a pressure of at least about 50 pounds per square inch.
[0116]
In one embodiment, the sheath fluid source is selected to be conditioned in both the pre-sorting and post-sorting sperm fluid environments, creating a chemically tailored sheath fluid environment for the cells. A sheath fluid source.
[0117]
In one embodiment, the chemically conditioned sheath fluid source comprises a solution comprising about 2.9% sodium citrate.
[0118]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, which includes the following:
  a. A cell source supplying cells to be analyzed by a flow cytometer;
  b. A sheath fluid source that creates a sheath fluid environment for the cells;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that acts to sort cells having the desired characteristics; and
  g. A collector with a buffer element,
Is provided.
[0119]
In one embodiment, the collector comprises a container comprising the buffer element.
[0120]
In one embodiment, the container comprises a wide collection tube.
[0121]
In one embodiment, the wide collection tube is at least about 15 mm wide.
[0122]
In one embodiment, the container comprises a test tube having the physical characteristics of the container adapted to flow.
[0123]
In one embodiment, the cell source comprises cells that are mechanically sensitive.
[0124]
In one embodiment, the cell source comprises sperm cells.
[0125]
In one embodiment, the collector comprises a buffer element.
[0126]
In one embodiment, the collector comprises a container comprising the buffer element.
[0127]
In one embodiment, the container comprises a wide collection tube.
[0128]
In one embodiment, the wide collection tube is at least about 15 mm wide.
[0129]
In one embodiment, the container comprises a test tube having the physical characteristics of the container adapted to flow.
[0130]
In one embodiment, the cell source comprises cells that are mechanically sensitive.
[0131]
In one embodiment, the cell source comprises sperm cells.
[0132]
In one embodiment, the nozzle, the oscillator, the cell sensing system, and the sorter identification system are part of a flow cytometer system, and the flow cytometer system comprises a high speed cell sorter.
[0133]
In one embodiment, the cell sorter sorts cells to be analyzed at a rate that sorts at least about 500 per second.
[0134]
In one embodiment, the high speed cell sorter is operated at a pressure of at least about 50 pounds per square inch.
[0135]
  According to another aspect of the invention, a desiredspermAn improved flow cytometer system for isolating cells is provided, the system comprising:
  a. A cell source supplying the cells to be analyzed by a flow cytometer;
  b. A sheath fluid source that creates a sheath fluid environment for the cells;
  c. A nozzle through which cells pass while subjected to a sheath fluid environment;
  d. An oscillator acting on the sheath fluid as it passes through the nozzle;
  e. A cell sensing system that responds to cells;
  f. A sorter identification system that acts to sort cells having the desired characteristics; and
  g. A collector comprising means for avoiding an impact between the cell and the collector,
Is provided.
[0136]
According to another aspect of the invention, a method for producing a mammal having a predetermined sex is provided, which method comprises the following steps:
a. Collecting sperm cells from a mammalian male species;
b. Determining the sexual characteristics of the plurality of sperm cells;
c. Sorting sperm cells according to the determination of their sexual characteristics;
d. Establishing a fertilized sample having a small number of sperm cells compared to a representative artificial fertilization dose;
e. Inserting at least a portion of a fertilized sample into a female female species;
f. Fertilizing at least one egg in a mammalian female species; and
g. Producing a progeny mammal of the desired sex;
Is included.
[0137]
In one embodiment, the step of collecting sperm cells from a mammalian male species comprises collecting sperm cells from a mammalian male species selected from the group consisting of cattle and horses.
[0138]
In one embodiment, the above steps of establishing a fertilized sample with a small number of sperm cells compared to a representative artificial insemination dose is provided in a representative unsexed artificial insemination event. Establishing a fertilized sample having no more than 10% of a representative number of sperm.
[0139]
  In one embodiment, the steps of establishing a fertilization sample having a small number of sperm cells compared to a representative artificial fertilization dose comprises 10ManyBovine fertilized sample of sperm cells, 250,000ofBovine fertilization sample of sperm cell, bovine fertilization sample of sperm cell of 300,000 or less, horse fertilization sample of sperm cell of 1 million or less, horse fertilization sample of sperm cell of 5 million or less, horse fertilization sample of sperm cell of 10 million or less And establishing a fertilization sample selected from the group consisting of horse fertilization samples of up to 25 million sperm cells.
[0140]
In one embodiment, the step of inserting at least a portion of a fertilized sample into a female of a mammalian species and the step of fertilizing at least one egg in the female of a mammalian species are each in a field environment. Achieved.
[0141]
In one embodiment, the step of inserting at least a portion of the fertilized sample into a female female of mammalian species further comprises the step of inserting the fertilized sample into the uterine horn by use of an embryo transfer device.
[0142]
In one embodiment, the step of determining the sexual characteristics of a plurality of sperm cells comprises staining the cells with a high concentration of dye.
[0143]
In one embodiment, the step of determining sexual characteristics of a plurality of sperm cells and the step of selecting sperm cells according to the determination of the sexual characteristics include the following steps:
a. Establishing a cell source supplying the cells to be sorted;
b. Chemically conditioning the sheath fluid to create a sheath fluid environment for the cells that is conditioned in both the pre-sort and post-sort cell fluid environments;
c. Sensing the characteristics of the cells;
d. Discriminating between cells having the desired sexual characteristics; and
e. Collecting cells having desired sexual characteristics;
Is included.
[0144]
According to another aspect of the invention, a method of producing a mammal of the desired sex is provided, the method comprising producing the mammal using the process.
[0145]
In one embodiment, the method further comprises the step of sorting the cells at high speed.
[0146]
  According to another aspect of the invention,spermA method for sorting cells is provided, which comprises the following steps:
  a. Establishing a cell source supplying the cells to be sorted;
  b. Chemically conditioning the sheath fluid to create a sheath fluid environment for the cells that is conditioned in both the pre-sort and post-sort cell fluid environments;
  c. Sensing the characteristics of the cells;
  d. Distinguishing between cells having the desired characteristics; and
  e. Collecting cells having desired characteristics;
Is included.
[0147]
In one embodiment, the method further comprises minimizing a chemical change that is provided as a result of subjecting the cell to the sheath fluid.
[0148]
In one embodiment, the step of establishing a cell source includes establishing a source of non-repaired cells.
[0149]
In one embodiment, the step of establishing a cell source includes establishing a source of sperm cells.
[0150]
In one embodiment, the step of establishing a cell source includes establishing a source of bovine sperm cells.
[0151]
In one embodiment, the step of establishing a cell source includes establishing a source of equine sperm cells.
[0152]
In one embodiment, the method further comprises fertilizing the mammal using a low dose of the sperm cell.
[0153]
In one embodiment, the step of chemically conditioning the sheath fluid to create a sheath fluid environment for the cells that is conditioned in both the pre-sort and post-sort cell fluid environments comprises about 2 Establishing a sheath fluid containing 9% sodium citrate.
[0154]
In one embodiment, the above step of chemically conditioning the sheath fluid to create a sheath fluid environment for the cells that is conditioned in both the pre-sort and post-sort cell fluid environments comprises a Hepes buffer. Establishing a sheath fluid comprising a crystallization medium.
[0155]
In one embodiment, the method further comprises the step of sorting the cells at high speed.
[0156]
In one embodiment, the step of sorting cells at a high speed includes subjecting the cells to a pressure of at least about 50 pounds per square inch.
[0157]
In one embodiment, the step of collecting cells having the desired characteristics includes buffering the impact of the cells with the collection container.
[0158]
In one embodiment, the step of buffering the impact of the cells with the collection container includes providing a wide opening in the container.
[0159]
  According to another aspect of the invention,spermA method for sorting cells is provided, which comprises the following steps:
  a. Establishing a cell source supplying the cells to be sorted;
  b. Establishing a sheath fluid to create a sheath fluid environment for the cells;
  c. Sensing the characteristics of the cells;
  d. Discriminating between cells having the desired sexual characteristics;
  e. Collecting cells having the desired sexual characteristics in a collector fluid; and
  f. Chemically conditioning the collector fluid to create an end collector fluid environment for the cells that is conditioned in the pre-sort fluid environment;
Is included.
[0160]
In one embodiment, the step of establishing a cell source includes providing an initial nutrient for the cell, and further comprising providing a collection fluid nutrient for the cell; And the above-described step of collecting cells having the desired characteristics in the collector fluid includes the step of equilibrating the initial nutrient and the collector fluid nutrient after completion of the step of collecting the cells.
[0161]
In one embodiment, the step of collecting cells having the desired characteristics in the collector fluid comprises establishing a citrate collection fluid comprising about 6% egg yolk prior to initiating this step of collection. To do.
[0162]
In one embodiment, the step of establishing a cell source includes establishing a source of bovine sperm cells.
[0163]
In one embodiment, the method further comprises fertilizing the mammal using a low dose of the sperm cells.
[0164]
  According to another aspect of the invention,spermA method for sorting cells is provided, which comprises the following steps:
  a. Establishing a cell source supplying the cells to be sorted;
  b. Establishing a sheath fluid to create a sheath fluid environment for the cells;
  c. Sensing the characteristics of the cells;
  d. Discriminating between cells having the desired sexual characteristics; and
  e. Collecting cells having desired sexual characteristics, comprising buffering the cells from colliding with a collection container;
Is included.
[0165]
In one embodiment, the step of buffering cells from colliding with the collection container includes providing a wide opening in the container.
[0166]
In one embodiment, the step of providing a cell source includes establishing a source of cells that are mechanically sensitive.
[0167]
In one embodiment, the step of establishing a cell source includes establishing a source of sperm cells.
[0168]
In one embodiment, the method further comprises the step of sorting the cells at high speed.
[0169]
In one embodiment, the step of sorting cells at high speed comprises subjecting the cells to a pressure of at least about 50 pounds per square inch.
[0170]
According to another aspect of the present invention, there is provided a method for producing a sex-separated sperm specimen, the method comprising producing a specimen using the above process.
[0171]
According to one embodiment, the method further comprises the step of sorting the cells at high speed.
[0172]
In one embodiment, the method further comprises providing a sex-specific sperm specimen for fertilizing a mammal using a low dose of the sperm cell.
[0173]
In one embodiment, the method further comprises providing a sex-identified sperm specimen for fertilizing the mammal using a low dose of sperm cells.
[0174]
According to another aspect of the invention, there is provided a method of producing a mammal of the desired gender, the method comprising producing a mammal using any of the processes described above.
[0175]
In one embodiment, the method further comprises the step of sorting the cells at high speed.
[0176]
In one embodiment, the method further comprises fertilizing the mammal using a low dose of sperm cells.
[0177]
In one embodiment, the method further comprises fertilizing the mammal using a low dose of sperm cells.
[0178]
According to another aspect of the present invention, there is provided a method for producing a plurality of sex-identified embryos from a female mammal, the method comprising the following steps:
a. Creating superovulation in a mammal to produce at least two eggs, comprising using an ovulation formulation to produce a plurality of eggs;
b. Determining the sex of male mammalian sperm cells;
c. Sorting according to the sex of sperm cells;
d. Inserting at least a portion of the sorted sperm cells into the uterus of a female mammal after initiation of ovulation; and
e. Fertilizing multiple eggs in the uterus to produce multiple sex-identified embryos;
Is included.
[0179]
In one embodiment, the above process of creating superovulation is facilitated during the estrous cycle.
[0180]
In one embodiment, the above step of using an ovulation preparation comprises injecting the ovulation preparation in half-day increments between any second and eighteen days of the estrous cycle.
[0181]
In one embodiment, the step of infusing the ovulation preparation in half-day increments comprises injecting with at least 7 injections, and further during the at least about the 6th and 7th injections, the ovulation manipulation system Including the step of incorporating
[0182]
In one embodiment, the step of inserting at least a portion of the sorted sperm cells into the uterus includes inserting sperm cells into both uterine horns of the uterus.
[0183]
In one embodiment, the step of inserting into both uterine horns includes the step of inserting sperm cells about 20-24 hours after initiation of the ovulation.
[0184]
In one embodiment, the above step of using an ovulation formulation to produce a plurality of eggs includes injecting a dose of follicle stimulating hormone multiple times per day.
[0185]
In one embodiment, said step of producing superovulation in a mammal to produce at least two eggs comprises supplementing said dose of follicle stimulating hormone with prostaglandin F-2-α. Further comprising the step of incorporating an ovulation manipulation system.
[0186]
In one embodiment, the step of injecting a dose of follicle stimulating hormone multiple times a day comprises 6, 6, 4, 4, 2, 2, 2 between 9 and 12 days of estrus. And injecting follicle stimulating hormone in about half a day increments at a dosage level of 2 mg, wherein the step of supplementing the follicle stimulating hormone dose with prostaglandin F-2-α comprises Injecting 25 mg and 12.5 mg of prostaglandin F-2-α at the sixth and seventh doses of stimulatory hormone, respectively.
[0187]
In one embodiment, the method comprises the following steps:
a. Staining sperm cells of a male mammal;
b. Sorting according to sex of sperm cells using high-speed flow cytometry; and
c. A step of concentrating the sorted sperm cells;
Is further included.
[0188]
In one embodiment, the step of inserting at least a portion of the sorted sperm cells comprises using a low dose of sperm cells.
[0189]
In one embodiment, the step of inserting at least a portion of the sorted sperm cells comprises using a low dose of sperm cells.
[0190]
In another aspect of the invention, a method of producing a mammal of the desired sex is provided, the method comprising producing the mammal using the process.
[0191]
In another aspect of the invention, a method of producing a mammal of the desired sex is provided, which method comprises producing a male and female identified sperm cell mammal using the process described above. And further sorting the cells at high speed.
[0192]
In one embodiment, the method further comprises fertilizing the female mammal using a low dose of sperm cells.
[0193]
In one embodiment, the method includes chemically adjusting the sheath fluid to create a sheath fluid environment for the cells that is conditioned in both the pre-sort and post-sort cell fluid environments. And the step includes establishing a sheath fluid comprising about 2.9% sodium citrate.
[0194]
In one embodiment, the above step of chemically conditioning the sheath fluid to create a sheath fluid environment for the cells that is conditioned in both the pre-sort and post-sort cell fluid environments comprises a Hepes buffer. Establishing a sheath fluid comprising a crystallization medium.
[0195]
In one embodiment, the method further comprises collecting the sex sperm cells and buffering the cells from colliding with the collection container.
[0196]
  According to another aspect of the invention,spermA method for sorting cells is provided, which comprises the following steps:
  a. Establishing a cell source supplying the cells to be sorted;
  b. Chemically conditioning the sheath fluid to create a sheath fluid environment for the cells that is conditioned by both the pre-sort and post-sort cell fluid environments;
  c. Sensing the characteristics of the cells;
  d. Discriminating between cells having the desired sexual characteristics; and
  e. Collecting cells having desired sexual characteristics;
Is included.
[0197]
In one embodiment, the method further comprises the step of sorting the cells at high speed.
[0198]
DETAILED DESCRIPTION OF THE INVENTION
As can be seen, the basic concepts of the present invention can be combined and encompassed in various ways. The present invention merely includes commercially practical low doses, intercourse fertilization and the results. For flow cytometry separation techniques, the present invention also includes both an improved flow cytometer system and a system for artificial fertilization and the generation of sex-specific sperm samples that can be used in animals produced by such techniques. Including. The present invention includes an overall process where a high success rate is possible even in a commercial environment. Further, since the techniques are disclosed in a general fashion, once the general principles are understood, the techniques can be applied to specific systems and applications. Although device enhancements are disclosed, it should be understood that these enhancements can be varied and combined in numerous ways, not just to achieve specific methods. Importantly, for all of the above, it should be understood that each of these aspects is encompassed by the present disclosure.
[0199]
As stated, the basic purpose is to separate X-bearing sperm from Y-bearing sperm. This is performed in a manner that isolates the two types of sperm so that each can be packaged and processed separately. Currently, this isolation is preferably performed through the use of flow cytometry. In general, flow cytometry is a well-understood technique. For example, its basic aspect is Cytomation, Inc. Are shown and discussed in various patents (eg, US patents and other publications listed above). Each of these patents and references cited herein is incorporated by reference; thus, one of ordinary skill in the art can readily understand the basic principles involved.
[0200]
In essence, flow cytometry includes the objects to be sorted (eg, cells) provided to the flow cytometer instrument through several types of cell sources. A conceptual device is shown in FIG. This flow cytometer device comprises a cell source (1) that serves to establish or supply cells or some other type of subject to be analyzed by the flow cytometer. The cells accumulate in the nozzle (2) in such a way that the cells are surrounded by the sheath fluid (3). This sheath liquid (3) is typically some sheath liquid such that the cell source (1) supplies the cells and the sheath liquid (3) is fed simultaneously through the nozzle (2). Supplied by a source (4). In this manner, it can be readily understood how this sheath fluid (3) creates a sheath fluid environment for the cells. Because the various fluids are provided to the flow cytometer at the same pressure, they exit the nozzle (2) and exit at the nozzle opening (5). By providing several types of oscillators (6) that can be controlled very precisely through oscillation control (19), the pressure wave is fixed in the nozzle (2) and at the nozzle opening (5). It can be converted into a fluid exiting the nozzle (2). Thus, the oscillator (6) acts on the sheath liquid (3) so that the flow (7) exiting the nozzle opening (5) eventually and in a controlled manner forms a drop (8). Since the cells are surrounded by a sheath fluid environment, the drops (8) may contain individual isolated cells or other objects within them.
[0201]
Because the drop (8) generally contains isolated cells, this flow cytometer distinguishes the droplets based on whether the appropriate cell or cells are contained within the drop, and Can be separated. This is achieved through the cell sensing system (9). This cell sensation system responds to cells contained within each drop (8) as discussed in the length of Larry Johnson, an original study by US Pat. No. 5,135,759 (not intended to be aligned) At least some types of sensors (10) are provided. As the Johnson patent describes sperm cells, this cell sensing system (9) is a relative presence or a relative non-existence of a specific dye that can be excited by several stimuli such as a laser exciter (11). It can produce effects that depend on its presence. Each type of sperm cell is stained with this dye, but the different lengths of the X and Y chromosomes result in different levels of staining. Therefore, by detecting the degree of pigment present in sperm cells, it is possible to distinguish between X-bearing sperm and Y-bearing sperm by their different luminescence levels.
[0202]
In order to achieve the ultimate separation and isolation of cells appropriate in flow cytometer separation technology, the signal received by the sensor (10) is fed into several types of sorter identification systems (12), which Can make a determination very quickly and can differentially charge each drop (8) based on the determination of whether the desired cells are present in the drop (8). In this manner, the sorter identification system (12) allows the electrostatic deflection plate (13) to deflect the drop (8) based on whether they contain suitable cells or other objects. Acts to allow. As a result, this flow cytometer acts to sort the cells by having the cells collected by one or more collectors (14). Thus, by sensing some properties of cells or other objects, the flow cytometer distinguishes between cells based on specific characteristics and places the cells in the appropriate collector (14). In the systems currently used to classify sperm, X-bearing sperm droplets are positively charged and thus deflect in one direction, Y-bearing sperm droplets are negatively charged and thus other Flow that is deflected in the direction and discarded (ie, unsortable cells) is not charged and is therefore collected in a suction tube or the like in the unbiased flow.
[0203]
Referring to FIG. 2, this process can be better understood. As shown in this figure, the nozzle (2) emits a flow (7) because the oscillator (6) (not shown in FIG. 2) forms a drop (8). Since the cell source (1) (not shown in FIG. 2) can supply sperm cells (15) that have been stained according to Johnson technology, light stimulation by the laser exciter (11) The presence or absence of charge on each drop (8) to separate from) is differentially determined by the sensor (10) so that it can be controlled by a flow cytometer. This control produces positively charged, negatively charged, and uncharged drops (8) based on their contents. As shown in FIG. 2, the particular drop is shown as a deflected drop (16). These deflected drops (16) are those that contain sperm cells of one sex or the other sex (15). They are then stored in a suitable collector (14) for later use.
[0204]
One aspect of flow cytometry that is particularly important for its application to sperm sorting is the fast operation of the flow cytometer. Advances are made under the MoFlo® trademark under Cytomation, Inc. In particular with a flow cytometer available via These flow cytometers have an extraordinarily increased sorting speed, thus making the flow cytometry a technology that appears to be a realistic commercial application for sperm sorting (other commercial applications). They act to achieve fast sorting at a significantly faster rate than another utilized classification. Specifically, Cytomation's MoFlo® flow cytometer operates at oscillator frequencies greater than about 5 kHz, and more specifically is operated within the range of 10-30, or even 50 kHz. obtain. Thus, droplets are formed at a very high frequency and cells contained within the sheath fluid environment can be emitted very quickly from the nozzle (2). As a result, each of the components that make up and part of the flow cytometer system, such as nozzle (2), oscillator (6), etc., can be set or selected to produce a fast cell sorter. In the application of a fast cell sorter for sperm cell sorting, sorting at a rate greater than about 500 per second is achieved. In fact, sorting speeds in the 1000 and 1200 range have already been achieved via high speed cell sorters. Importantly, the term “fast” is a relative term such that other benefits and specific applications in flow cytometry are achieved, and the aspects considered “high” can vary or are absolute It should be understood that this can remain the same. In either definition, this general principle is that when sorting specific cells such as sperm cells, this classification occurs at a rate that the flow cytometer parameters and physical properties are significant relative to the cells themselves. Is to get.
[0205]
One aspect of high speed sorting that appears to be effective when sorting sperm cells via flow cytometer separation techniques is that of pressure and other stresses where sperm cells are subjected to flow cytometers. For example, when operated at high speed (and an alternative definition of “high speed”), the flow cytometer may be operated at a pressure of 50 pounds per square inch and more than 60 pounds per square inch. These pressures can be considered high. This is because these pressures can have an effect on the cells being sorted. The clue as disclosed in this invention for this aspect is the fact that the stress threshold of a particular cell is a determinant. Furthermore, as further knowledge is gained, the stress threshold can be shown to be a function of the combined effect, such as the specific species of cells, or the specific pre- or subsequent handling of the cells. The clue in this regard is that the stress imposed on the cells can actually change their viability and their ability to achieve the desired result. In the case of pressure, simply subjecting sperm cells to higher pressures as a result of operation of the flow cytometer at that pressure can result in reduced performance of the cells. In one respect, the present invention acts to minimize these stresses, thus producing greater efficiency as discussed below, as well as lower doses.
[0206]
In considering aspects of cellular stress, the present invention operates in a manner that minimizes this stress. These stresses can be minimized at any point throughout the cycle or the entire process by which the animal is controlled, screened or fertilized. Importantly, the stress imposed by handling cells in the flow cytometer appears to be significant for this application. In one embodiment of the invention, the sheath fluid may be specifically selected and provided in a prepared manner in both (or either) the pre-sorting cell fluid environment or the post-sorting cell fluid environment. In nature, it is possible to adjust either the pre-sorting fluid or the post-sorting fluid, but in one embodiment, the present invention regulates the sheath fluid (3) so that its cells than previously achieved. Imposes significantly less stress on In one respect, the present invention is significant in that it removes the total focus from the focus of the flow cytometer's operation relative to the focus in handling and removing stress from the cells themselves. For example, while it is known to utilize fluids with appropriate pH factors or osmolarity, the present invention may exist for certain chemical compositions in which cells can be over-responsive. Recognize that. These hyperresponsive chemical compositions can vary naturally, on a cell basis, or even before cell handling. Importantly, it is now apparent that certain metabolic chemical compositions of sperm cells (eg, citrate) appear to prevent abnormally high stress on the cells. Thus, hyperresponsive chemical compositions can be defined as chemical compositions in which cells are particularly responsive within the context of their functionality and existing handling techniques. Regarding sperm cells, it is clear that metabolic compositions, in particular citrate stability for bovine sperm cells and hepes buffer stability for horse sperm cells, can be very important. Thus, the present invention acts to minimize changes through the choice of type of operation or substrate, which can serve as a means for minimizing the changes experienced by cells.
[0207]
For the sheath fluid, the material can be selected according to one embodiment of the present invention and consequently chemically adjusted to give minimal change. Thus, by selecting an appropriate sheath fluid not only within the context of the flow cytometric parameters, but rather within the context of the cellular parameters themselves, the changes experienced by the cells and the results across this sort can be enhanced. This is conceptually illustrated in FIG. FIG. 3 shows several types of chemical factors (eg, citric acid or other factors). This is because it can exist throughout the various phases of the process. For example, the four phases shown represent, but are not limited to, flow cytometry separation techniques: Phase I can represent the presence of cells in the cell source (1), and Phase II can be in a sheath fluid environment. May indicate the presence of cells as sorted in, phase III may indicate cells such as cells collected after sorting, and phase IV may indicate cells reconstituted in the storage medium after sorting. These four phases as shown in the prior art can experience very different chemical factor environments. However, as conceptually shown in the present invention, cells can undergo very small changes, most notably the dip or drop experienced between this phase I and phase II is substantially present. I can't. This is as a result of selection of an appropriate sheath fluid as described above. Thus, as a result of being subjected to an appropriate sheath fluid, the cells in the present invention may experience very low levels of stress.
[0208]
One of the universalities that may exist with respect to this phenomenon is the fact that certain chemical compositions can represent chemical compositions that are more responsive than others. In nature, this can vary depending on the sperm species, this treatment, or even the type of cells involved, but cell viability for their intended purpose (here artificial fertilization) is large, Varying, either natural or sorted or both, the cells appear to exhibit characteristics that are overresponsive with respect to their chemical composition. By choosing a particular metabolic chemical composition, the most significant citric acid or chemical within the citric acid cycle appears to be capable of great advantages. Thus, for bovine sperm applications, this sheath fluid (3) is selected and adjusted to present about 2.9 percent sodium citrate composition. Specifically, a 2.9 percent sodium citrate solution can be made as follows:
1. 29.0 g of sodium citrate dihydrate (Na_ThreeC₆H_FiveO₇・ 2H₂O)
a. Dissolve sodium citrate in 3/4 water batch, then add water to volume
2. Add deionized or Nanopure water to a final volume of 1,000 ml.
3. Transferred to a bottle and 15 lbs pressure (245^oAutoclave at F) for at least 30 minutes.
[0209]
a. Autoclave the solution (loose cover) using conditions that minimize evaporation.
[0210]
b. Note that the water does not evaporate.
4). Cool slowly at room temperature.
Store sealed in a cold room at 5.5 ° C.
Further, the sodium citrate solution can be filtered for the sheath liquid.
6). Filter through a 0.22 micron filter using aseptic technique.
[0211]
Interestingly, for horse sperm cells, such compositions do not perform as well. Rather, for horse sperm cells, hepes buffered medium, such as hepes bovine gamete medium—especially J. J. et al. HBGM3-, previously made by Parrish, works. This medium is discussed in the paper “Capacitation of Bovine Sperm by Heparin”, 38 Biology of Reproduction 1171 (1988), incorporated herein by reference. This is not only a surprise because it is not the same type of material used for bovine sperm, but the actual buffer was also originally developed for bovine applications. Accordingly, in the application of horses, a sheath fluid containing a hepes buffer is selected. This solution may have a pH of about 7.54 at room temperature (pH = 7.4 at 39 ° C.) having the following composition:
[0212]
[Table 1]

One other aspect that can interact in the present invention is the fact that the cells involved can experience abnormal sensitivity. In one respect, this may be due to the fact that sperm cells are within the class of cells that are non-repaired cells. That is, they do not have the ability to repair themselves, so they may need to be treated more sensitively than is typical for flow cytometers or other handling equipment. . Thus, this enhancement may be particularly applicable when a flow cytometer, or other separation device, acts to establish a source of sperm cells. Another possible related aspect that may be unique to a class of cells such as sperm cells is the fact that their DNA is non-repaired, non-replicating, and non-transcribed. Any of these factors can be effective, and they can be related either individually or together. Thus, the teachings of the present invention can be applied to all gamete cells or even viruses, which are non-repaired, non-translated, non-transcribed cells.
[0213]
Another aspect of flow cytometer processing that may also be important is the fact that the cells are properly treated both chemically and physically after sorting. As shown in FIG. 4, in order to acquire the cells in the drop (8) into the collector (14), the containers that make up the collector are appropriately sized so that there is no gap between the cells and the container itself. It can be important to act as some means to avoid impact. Although it is known to place an initial collector fluid (17) at the bottom of the container that collects the cells so that the cells do not hit the bottom of the container, the presentation of flow due to the collision of the cells into the container As well, it is clear that the simple breadth of the container to handle variations in unavoidable splashing can be used to enhance this result. In one respect, it can act as a buffering element so that cells that can be mechanically sensitive, i.e. cells that can be destroyed or damaged by impact, can be treated appropriately. Thus, when the cytometer source establishes cells that are physically delicate cells, such as cells to be sorted, the size of the opening (18) of the container in a manner that avoids contact with the cells. It may be important to provide some type of cushioning element, such as a wide collection tube, that serves to place the wall. Therefore, since this tube does not exist so close to the side wall, there is any significant chance of contact between those cells to be sorted and the tube wall. In this manner, it is desirable to have a similarly wide collection tube in addition to the collector fluid (17). Perhaps it may be significant to simply provide a wide opening in the container that serves as part of the collector (14). It has been found that it would be significant to provide a container having an opening with an internal diameter of at least 15 millimeters for applications utilizing rapid classification of sperm cells. Specifically, when utilizing 14 ml Falcon tubes in such applications, minimal physical damage to the cells has been found as a result of the collector (14).
[0214]
It should be noted that even a 14 ml Falcon tube may not be optimal. In particular, it is believed that it may be best to design a collection vessel that is suitable for the flow geometry (ie, a “stream-matched container”). This flow-compatible container may have any or all of the following features: a relatively wide opening, an oval shaped opening, a smaller height than currently involved: width ratio, angle Appearances that are marked or otherwise adjusted to show side walls parallel to the falling flow. It is also desirable to provide a movable element such as a ball bearing or a mounting element such as a medium to adapt the variable orientation of the test tube to the falling flow desired to be collected. Furthermore, the physical characteristics of the classification of containers such as existing test tubes (described as “Falcon” test tubes) include not only the width of the test tube but also the material from which the test tube is made (eg, cell May also be mentioned. (These material choices are well known for 14 ml Falcon tubes.) Thus, the container and its collection fluid can also act as a buffering element to minimize physical damage to the cells. This may also work to facilitate the collection of the appropriate number of sperm, depending on size, without significant dilution effects.
[0215]
Another aspect of the collector fluid (17) may be the fact that it can also act to minimize chemical stress on the cells. In one respect, it can be important to provide nutrients to the cells both before and after sorting, so the collector level (17) can be chosen to provide a regulated level of nutrients, so this nutrient level is It is equilibrated both before and after sorting. For bovine sperm where egg yolk citrate nutrients are utilized at the 2% egg yolk level, good results are obtained using the 6% egg yolk citrate level (ie, 6% egg yolk content in the citrate solution). Was found to provide. This is a result of the capacity existing before and after the sorting event. The collector liquid (17) can be started (before sorting) with a volume of about 2 ml. A sorting event can add approximately twice this volume (ending at 3 times the initial starting volume) and yolk in solution (due to clogging and other flow cytometer considerations). There is little citrate. Thus, in terms of the level of the amount of egg yolk citrate present, the final result may be equal to the 2% egg yolk citrate content in the citric acid solution due to the starting result, ie the volume contained. Accordingly, the collector liquid (17) may be selected to create a final collector liquid environment that is equilibrated with the initial nutrient or other fluid environment. In this manner, the collector fluid can act to minimize changes in the time and level of composition that the cells are subjected to. Of course, these fluid environments may be shown in the flow cytometer or may exist at some other important time, and the important point is that the cell is at any point in the life of the cell. It is to minimize the applied stress. In addition, since the initial chemical amount can be varied (eg, the percent egg yolk content in citrate can be varied up or down), similarly, the starting collection fluid environment or various volumes also have the same end result Can be varied. Thus, before starting the sorting process, the collector fluid is present at a 6% egg yolk content in the citrate solution, and after the sorting event has ended, the collector fluid (with sex-specific sperm) results in It can be 2% egg yolk content in citrate solution as well as the initial nutrient content.
[0216]
In the latter use, these sperm cells can be processed for a 20% egg yolk content in citrate solution for other reasons. However, these changes are not recognized as providing stress to the cells as they are only a known part of the overall fertilization process. Of course, this level can be easily understood by those skilled in the art and can be varied, but a 20% egg yolk citrate buffer can be constructed as follows:
I. Final composition:
80% sodium citrate solution (72 mM)
20% (volume / volume) egg yolk
II. Preparation for 1L:
A. Sodium citrate solution
1. 29.0 g of sodium citrate dihydrate (Na_ThreeC₆H_FiveO₇・ 2H₂O)
2. Add deionized water or Nanopure water to a final volume of 1,000 ml
3. Transfer to bottle and autoclave at 15 lbs pressure (245 F °) for at least 30 minutes
a. Autoclav the solution (loosely cap) using conditions to minimize evaporation
b. Be careful not to let water boil and leak out
4). Cool slowly at room temperature
5. Keep tightly sealed and store in a low-temperature room at 5 ° C.
B. Egg preparation
1. Get fresh eggs from good commercial sources
2. Wash the eggs to avoid dust (do not use excess surfactant) and rinse
3. Immerse eggs in 70% ethanol for 2-5 minutes
4). Remove eggs, dry (or wipe dry) and store on clean towels
C. Preparation of extender
1. Use clean sterile glassware
2. A-fraction (fraction without glycerol)
a. Place 800 ml of 2.9% sodium citrate solution in a 1,000 ml graduated cylinder
b. Antibiotic levels for the extender-free glycerol fraction (A fraction) can be as follows:
i. Tylosin = 100 μg / ml
ii. Gentamicin = 500 μg / ml
iii. Linco-Spectin = 300/600 μg / ml
c. Add 200 ml of fresh egg yolk as outlined below (section D)
i. Mix thoroughly
d. This provides an A fraction extender based on 2.9% sodium citrate with a concentration of antibiotic known to be non-toxic to 20% egg yolk and bovine sperm.
e. The extender can be stored at 5 ° C. overnight.
[0217]
f. Decant the supernatant (upper 800 ml) the next day
g. Warm to 37 ° C before use the next day
D. Complete the following procedure to add egg yolk to the buffered solution
1. Thoroughly wash and clean the eggs (see B above)
2. Divide the egg and separate the egg white and egg yolk using an egg yolk separator. Alternatively, the yolk is put in and out of the egg shell alternately two or three times. Do not break the yolk membrane
3. Place egg yolk on 15cm sterile filter paper
4). Place the filter paper on the graduated cylinder containing the buffer, squeeze the yolk (breaking the yolk membrane) and drain it from the gold-treated filter paper to the graduated cylinder. Typically, about 12-15 ml of egg yolk can be obtained from one egg.
[0218]
Another aspect that can interact with the various factors of the present invention is the use of low doses of sperm for artificial insemination and the like. Further background on the aspect of artificial insemination, which has been identified, is described in “Prospects for Sorting MammalianSperm”, Rupert P. et al. Amman and George E.M. Seidel, Jr. , Colorado Associated University Press (1982), incorporated herein by reference. As mentioned, natural fertilization involves billions of sperm counts. Typical artificial fertilization is currently performed on millions of sperm for bovine species and on hundreds of millions of sperm for horse species. By the term “low dose”, the dose of sperm used in a fertilization event is less than half of the number of representative sperm provided in a typical artificial fertilization event, or preferably less than about 10% of it. Even means that there is. Thus, the term “low dose” is to be considered in the context of a typical artificial fertilization dose, and also as an absolute number. For bovine sperm, which currently provides 1 million to 10 million sperm, the low dose process can be considered an absolute number of about 500,000 sperm counts, or perhaps less than 300,000 sperm. In fact, the success of artificial fertilization at a good rate by utilizing the technology of the present invention has been reported in the paper “Uterine Horn Insulation of Heifers With Very Low Numbers of Non-Srozen of Non-Frozen 1255” (1997). This was shown at the fertilization level of 100,000 and 250,000 spermatozoa (pregnancy rates of 41% and 50%, respectively) as shown in this document, which is incorporated herein by reference. Since sperm cells appear to be sensitive to dilution, these results may show particular interdependencies for the use of low dose sperm samples for the various techniques of the present invention. The absolute number can be species dependent. For equine species, a low-dose process can be envisaged even if less than about 25 million, 10 million, 5 million, or 1 million sperm.
[0219]
Another aspect that may be important is the fact that sexual identification of sperm is utilized in artificial insemination systems by the (or other) technique of the present invention. Thus, with regard to flow cytometer technology, the technology of the present invention may be particularly relevant when using a collector (14) to provide sperm for artificial insemination. Furthermore, the combination of both the use of artificial insemination and use in a low dose environment can both be synergistic, which can make the various techniques of the present invention particularly suitable. Of course, sex-separated spermatozoa can be utilized not only in artificial fertilization aspects but also in other techniques such as in vitro fertilization techniques.
[0220]
The process of collecting, selecting, and actually artificially fertilizing animals through the use of flow cytometry or other separation techniques involves various steps. In the situation of bovine artificial insemination, first, semen is collected from the cow using an artificial vagina. This is a rate of about 1.5 billion sperm / ml. This raw semen can be checked using a spectrophotometer to assess concentration. This semen can then be evaluated microscopically to ensure that appropriate motility and viability criteria are met. Antibiotics can then be added. As a result, the first sample may have about 60-70% forward mobility per ejaculation. For processing steps, sperm counts can be obtained at a manageable level of about 100 million / ml (for flow analysis) using some type of TALP (Tyrode Album lactate pyruvate) dilution. TALP not only nourishes sperm cells, but can also enhance sperm for the staining process. Prior to staining, some species, such as horse species, can be centrifuged. The staining process can be performed according to multiple staining or single color staining protocols, the latter being the Johnson patent and related technology. The dyeing process can be performed while also adjusting the extender to create a suitable nutrient environment. In bovine applications, this involves adding about 20% egg yolk content into the citrate solution immediately after staining. Furthermore, it has been discovered that in staining sperm cells, better results are achieved with higher staining amounts than can be expected to a certain extent. This may include the step where the high density dyeing step uses a dye amount of tens of μM content (eg, discussed in the examples below where Hoechst 33342 dye is used at a 38 μM content).
[0221]
As an incubation period after adding the dye, for example, a step of incubating at 34 ° C. for 1 hour to promote dye uptake at a concentration of about 100 million sperm cells / ml can be used. They can then be filtered to remove sperm cell aggregation. A dilution or expansion to about the desired sorting concentration of about 100 million sperm cells / ml can then be performed. A sorting step according to the various techniques discussed above is then performed, from which sperm cells can be collected in the collection phase. As previously mentioned, collection can occur with samples having about 2% egg yolk citrate concentration content (for bovine species). The sample can then be concentrated to about 3-5 million sperm cells / ml by centrifugation after the sheath and preservation solutions can be removed. Final expansion can then be achieved with either 20% egg yolk citrate or Cornell Universal Extender or the like. Cornell Universal Extender can have the following composition for 1000 ml:
14.5g Sodium citrate dihydrate
2.1g NaHCO_Three
0.4g KCl
3.0g glucose
9.37 g glycine
0.87g Citric acid
For a 20% egg yolk composition, 800 ml of the above preparation and about 200 ml of egg yolk may be used.
[0222]
After this last expansion, 3-5 million sperm / ml (for bovine species) can occur. The sample can then be cooled to slow down sperm metabolism and to allow use over a longer period of time. In horse species, this sample can then be used in the fallopian tube process or other fertilization processes, as is well understood by those skilled in the art. For bovine sperm, the sample can be diluted once more to the desired dosage level. It has been found that since dilution can affect sperm cell viability, it may be appropriate to avoid having the level of dilution become too large by providing a smaller sample. Nevertheless, of the separation techniques used, currently a low dose of about 300,000 sperm / 0.184 ml can be achieved. In addition, it may be desirable to maintain seminal plasma levels at a level of about 5%, but the consequences of this need are now tied. The sperm cell sample can then be placed in a straw for use in artificial insemination and transported to a calf or heifer for fertilization.
[0223]
In order to achieve artificial fertilization at a convenient time, the estrous period of heifers or heifers can be determined according to techniques well known in the art by using prostaglandin F2_αCan be synchronized by known techniques such as This latter content is described in the paper “Prostaglandin F2_α-A Fertility Drug in Dairy Cattle? 18 Therogenology 245 (1982) (which is incorporated herein by reference) was reported to potentially achieve enhanced fertility in heifers. Can be particularly valuable in terms. In recent results, the foregoing has not been maintained, but the present invention may be to demonstrate extra viability in the context of low-dose fertilization with sex discrimination. Then, for bovine species, artificial fertilization can be achieved by use of an embryo transfer device that places sperm cells deep within the uterine horn. This can be achieved at some later time, such as 12 hours after the peak, rather than at the peak typically used in artificial insemination. This is because fertilization for male and female identified artificial fertilization can occur somewhat later. Utilization of embryo transfer equipment can be used because the uterine wall can be very sensitive with respect to such low dose male and female discriminated fertilization.
[0224]
Furthermore, this technology can be combined to achieve even more efficient production. In particular, each of the processes just invented that allows high-speed sorting and low-dose artificial insemination of male and female identified embryos is feasible in superovulated animals. Superovulation can be achieved by the use of a superovulatory pharmacological agent or any other technique. An ovulation inducing agent can act directly or indirectly, for example, by a series of reactions to achieve more egg production than normal. The combination with superovulation is surprising because it has previously been found that superovulation prevents such a combination. Because sperm transport is impaired in superovulated cattle, animals were frequently artificially inseminated on multiple occasions and / or with multiple doses of semen. Also, previous procedures for sexing semen were relatively slow; thus, using a combination of these new techniques, a superovulation-inducing agent (eg, FSH (follicle stimulating hormone)) treated cattle It is interesting to determine the fertilization rate after a single artificial insemination using a total of 600,000 sexed unfrozen spermatozoa.
[0225]
Illustratively, 12 Angus cross heifers were superovulated using standard procedures: 6, 6, 4, 4, 2, 2, 2, and 2 mg of FSH on day 9 of the estrous cycle Intramuscular injections were made at half-day intervals beginning between eyes 12 and 12; 25 mg and 12.5 mg of prostaglandin F-2α were injected intramuscularly with the 6th and 7th FSH injections. Sperm from cows of unknown fertility were stained with Hoechst 33342 and then screened using a MoFlo® flow cytometer / cell sorter to obtain 700-800 live sperm at each sex / second. It was. The average sorting purity was 89% of the desired sex. The sorted sperm is cooled to 5.degree. C. and centrifuged at 650 g for 10 minutes to 3.36 × 10.⁶Concentrated to sperm / ml and stored for 4 hours. 184 μl is then placed in a 0.25 ml plastic straw; half of this dose is artificially fertilized at each uterine horn 20 to 24 hours after estrus using an automatic side-opening embryo transfer sheath. did. Embryos were collected 7 or 16 days after estrus by standard non-surgical procedures. The results were similar between the collection on day 7 and 16 and with X and Y sorted sperm. Embryos were collected from 9 heifers. There were 52 embryos (average 4.3 ± 5.3 / donor), 13 delayed embryos and 31 unfertilized eggs at the normal stage of development. Forty-six embryos were sexed by PCR using primers for Y-chromosome-specific DNA sequences; 43 (93%) were of the intended gender. This study only involved a small number of animals, but surprisingly, artificial insemination of superovulated treated heifers with only a total of 600,000 (viable) male and female identified unfrozen spermatozoa Gave similar results to the conventional procedure. The above variations can also be achieved with (including, but not limited to) a step other than by flow cytometer means, a step of achieving superovulation in other ways, a step of increasing fertility in other ways, etc. Can be done.
[0226]
Furthermore, the harmonization of the method of discriminating males and females based on DNA content, high-speed flow cytometer / cell sorter, and procedures for artificially fertilizing heifers with fewer than 500,000 total sperm without compromising fertility, Within only a few years, the potential for a semen industry that identified viable sexes in cattle was raised. There are many applications for sperm that have been sexed with greater than 85% accuracy. Perhaps most obvious is the artificial insemination of cows in one subset (both dairy and beef cattle) and the reverse subset (both dairy and beef cattle) produced for meat to replace a group of females. Crossing with a completely different type of cattle. A very important subset of the above is the production of cows by artificial insemination of heifers with X chromosome-bearing sperm. This is mainly due to the smaller size, resulting in a lower incidence of dystocia than bulls. In addition, providing young dairy cattle is far more effective in favoring heifers. Having on average more than 85% heifers also allows them to manage dairy cows, on average they are less than two live cattle per life. This is attractive for reducing problems associated with pregnancy and labor. A single sex system for meat production is also possible. Here, because each female replaces itself and is slaughtered for 2-3 years, the proportion of using and maintaining much higher nutrients in the system for growth is lower. Sex-identified semen is useful for in vitro artificial fertilization and for artificial fertilization of superovulated cattle for embryo transfer. Often, despite the fact that one sex cow is much more valuable than the other, and accurate methods are available to discriminate embryos, they are time consuming and of the generated embryos Half is sex that is not so valuable. Accurately sexed semen is presumed to be widely compatible with bovine artificial insemination if the additional fee for sex discrimination is low and fertility is minimally compromised. The proportion of beef cattle that are artificially inseminated will probably increase in beef cattle with semen that is substantially sexed.
[0227]
Interestingly, better results can be achieved by fertilization deep into the uterine horn than fertilization into the uterine body where such fertilization is normally placed. Perhaps it is also surprising that samples so much studied in this way show no difference between ipsilateral and contralateral fertilization when achieved deep within the uterine horn. is there. By being deep, it should be understood that the insertion is well placed in the uterine horn using an embryo transfer device. The fact that the results do not appear to differ significantly using ipsilateral and contralateral fertilization has led the inventors to propose the use of both fertilizations, so that appropriate The process of identifying uterine horns can no longer be necessary.
[0228]
As a result of fertilization, it is of course desirable to produce an animal of the desired sex. This animal can be produced according to the system discussed above through the use of sexed sperm preparations. It should also be understood that the techniques of the present invention may find application in other techniques such as, for example, laparoscopic fertilization, fallopian tube fertilization, and the like.
[0229]
As an example, the following experiment was conducted. Rather than using all of the aspects of the invention described herein, they show possible performance enhancements through different aspects of the invention. In addition, a summary of some experiments is included in the previously cited paper “Uterine horn fertilization of heifers using very few unfrozen and male and female sorted sperm”. This paper summarizes some of the data showing the effectiveness of the present invention. The experiment was performed using male and female sorted non-frozen sperm cells and was highly successful as follows.
[0230]
【Example】
Example 1
Angus heifers (13-14 months old (mo) and moderate physical condition) were synchronized with 25 mg prostaglandin F-2α at 12-day intervals and upright estrus Fertilization was performed 6-26 hours after the standing estrus was observed. Semen freshly collected from 3 bulls 14-26 months old was collected in TALP medium for 1 hour at 34 ° C., 75 × 10⁶Incubated in 38 μM Hoechst 33342 at sperm / ml. Sperm were used from a laser excitation at 351 nm and 364 nm at 150 mW using a MoFlo® flow cytometer / cell sorter operating at 50 psi and 2.9% Na citrate as the sheath fluid. Sorted by sex chromosomes based on epifluorescence. Sperm with X chromosomes (approximately 90% purity as confirmed by recovering sonicated sperm aliquots) at approximately 500 live sperm / sec and containing 100 μl Cornell Universal Extender (CUE) 2 Collected in a 20 ml Eppendorf tube with 20% egg yolk. The collected sperm is centrifuged at 600 × g for 10 minutes and 1.63 × 10 6⁶Resuspended in CUE with raw sperm / ml. For control of semen that has not been sexed; Hoechst 33342-stained sperm is 9 × 10 9 in sheath fluid.^FiveDilute to sperm / ml and centrifuge and 1.63 × 10 6 in CUE⁶Resuspended in progressively motile sperm / ml. Male and female sorted semen and liquid control semen were cooled to 5 ° C. for 75 minutes and loaded into a 0.25 ml straw (184 ul / straw). The straw was transferred to a temperature controlled beverage cooler 240 km at 3-5 ° C. 5-9 hours after sorting for fertilization. Male and female sorted semen and fluid control semen were fertilized using side-opening blue sheaths (IMV) and half of each straw was placed in each uterine horn (3 × 10^Five(Raw sperm / heifer). As a standard control, semen from the same bull was frozen in a 0.5 cc straw according to standard procedures (average 15.6 × 10 6⁶Motility sperm / post-lysis dose), lysed at 35 ° C. for 30 seconds and fertilized into the uterus. Treatments were averaged for 3 bulls and 2 fertilizers at a ratio of 3: 2: 2 fertilization for male and female screened semen and 2 controls. Pregnancy was determined by ultrasound 31-34 days after fertilization and confirmed 64-64 days after fetuses were also sexed (blindly). The data is shown in the table.
[0231]
[Table 2]

^{a, b}The sex selection ratio for values with different superscripts is different (p <0.02).
[0232]
The pregnancy rate with sex-sorted semen was only 80% of the control, but this difference was not statistically significant (> 0.1). A single pregnancy is lost between 64 and 67 days in each sexed and frozen control group; 18 (95%) of 19 fetuses in the sexed group are female and 30 in the control group Of these, 20 (67%) were female. Liquid semen control yields substantially the same pregnancy rate as frozen semen control, which contains more than 50 times more motile semen (more than 120 times the total sperm), which means that the Illustrates the effectiveness of dose fertilization. We have significantly changed the sex ratio in cattle using flow cytometer technology and artificial insemination.
[0233]
Similarly, one experiment was performed using unfrozen, unfrozen sperm cells and could be reported as follows.
[0234]
(Example 2)
The purpose of the experiment was to determine the pregnancy rate when fertile fertile cows were used with an extremely small number of frozen spermatozoa under ideal field conditions. Semen from 3 Holstein bulls, with the above average pregnancy rate, was added 2 × total sperm per 0.25 ml French straw in homogenized milk, 7% glycerol (CSS) extender, and 5% homologous semen. 10^Five5 × 10^FiveOr 10 × 10⁶(Control) and transferred to liquid nitrogen vapor and frozen. Semen was thawed in a 37 ° C. water bath for 20 seconds. 13-15 months old Holstein heifers weighing 350-450 kg are infused twice with 25 mg prostaglandin F-2-α (Lutalyse®) at 12 day intervals, and Fertilization is performed using an embryo transfer straw gun and a side-opening sheath, and half of the semen enters deep into each uterine horn 12 or 24 hours after detection of estrus. Experiments were performed in 5 replicates over 5 months and averaged over 2 fertilizers. Care was taken to keep the fertilizer warm because the ambient temperature for breeding was often -10 ° C to -20 ° C. Pregnancy is determined 40-44 days after estrus by detection of viable fetus using ultrasound and confirmed 55-62 days after estrus; 4 of 202 conceptions are Lost at the time of. In 55-62 days, pregnancy rate is 2 × 10^Five5 × 10^FiveAnd 10 × 10⁶The total sperm / fertilization (P <0.1) was 55/103 (53%), 71/101 (70%) and 72/102 (71%). Pregnancy rates differed between bulls (59, 62, and 74%, P <0.05), but between experimenters (64 and 65%) or after fertilization (at 12 hours) 65%, 64% in 24 hours, N = 153, each time) was not different. Using the described method, the pregnancy rate in heifers is 5 × 10 5 per fertilization^FiveAnd 10x10⁶Similar to using total sperm.
[0235]
One experiment was also performed on male and female sorted non-frozen sperm cells and can be reported as follows:
(Example 3)
Semen was collected from Atlantic Breeders Cooperative bulls, diluted 1: 4 with HEPES buffered extender + 0.1% BSA and transported to Maryland, Beltsville for 160 km (about 2 hours). The semen was now screened by flow cytometry in a TEST yield (20%) extender using the method previously described (Biol Reprod 41: 199) at ambient temperature. 2 x 10 of each sex per 5-6 hours at about 90% purity⁶The sorting speed up to sperm was achieved. Sperm were centrifuged at 300 x 4 min for 2 x 10⁶Concentrated to sperm / ml. Some sperm were sorted into extenders containing homologous semen (final concentration 5%). Sorted sperm are transported by plane to Colorado (approximately 2,600 km) and stored at either ambient temperature or 5 ° C. (Equitainer (shielded device with ice compartment) for more than 6 hours) During cooling). 11-36 hours earlier than estrus was detected, heifers or dairy cows were fertilized within 9-29 hours of the end of the sperm sorting session. Sperm (1-2 x 10 in 0.1 ml^Five) Was placed deep in the uterine horn on the ipsilateral side of the ovary with the largest follicle determined by ultrasound during fertilization.
[0236]
None of the 10 females became pregnant when fertilized with sperm that had been transported and stored at ambient temperature. Of the 29 females that were fertilized with sperm cooled at 5 ° C. during transport, 14 were pregnant for 4 weeks and 12 (41%) for a gestation period of 8 weeks. Eleven of the 22 animals fertilized within the last 10 hours of screening became pregnant for 8 weeks, but only one of the 7 animals fertilized 17-24 hours after screening became pregnant. There was no significant effect of adding seminal plasma. One of the 12 fetuses is not the predicted gender, one is ambiguous, and 10 is the predicted gender as determined by 60-70 day ultrasonography of pregnancy. there were.
[0237]
Subsequently, 33 additional heifers were fertilized with 0.05 ml (semen spread as described above) in each uterine horn without the use of ultrasonography; only 3 cases were post fertilized She became pregnant for 4 weeks and only one patient remained pregnant for 8 weeks. However, using different bulls from the previous group and all fertilizations were performed 18-29 hours after selection. Another 38 bulls from our laboratory were similarly fertilized using selected sperm from another bull (about 22 hours after selection); any of these Did not get pregnant for 8 weeks after fertilization.
[0238]
In summary, it is possible to achieve pregnancy in cattle by artificial insemination of sperm selected for sex chromosomes by flow cytometry, and fetal sex ratios are reanalyzed for the DNA content of the selected sperm Approaches the sex ratio predicted by (90%). However, pregnancy rates varied greatly in these preliminary experiments that required long-distance sperm transport. The fertilization rate decreased sharply 17 hours after selection, but there was some confusion. Because different bulls were used at different times. Further studies have been conducted to determine whether the variation in pregnancy rate is due to bull differences, fertilization techniques, due to the interval between selection and fertilization, or due to other factors. is needed.
[0239]
Finally, one experiment was also performed with unfrozen, unfrozen sperm cells and can be reported as follows.
[0240]
Example 4
The purpose of this example was to determine the pregnancy rate when heifers were fertilized with very few sperm under ideal experimental conditions. Sperm from 3 Holstein bulls were aliquoted in 1 × 10 6 in Cornell Universal Extender and 5% homogeneous semen.^FiveOr 2.5 × 10^FiveSperm / spread to 0.1 ml; 2.5 × 10⁶Total sperm / 0.25 ml was used as a control. Fully spread semen was packed into a modified 0.25 ml plastic french straw to deliver a fertilization dose of 0.1 or 0.25 ml. Semen was cooled to 5 ° C. and used 26-57 hours after collection. Holstein heifers 13-15 months old, weighing 350-450 kg, injected with 25 mg prostaglandin F-2α (Lutalyse®) at 12-day intervals and 24 hours after detection of estrus A single uterine horn was fertilized using an implanted straw gun and a side opening sheath. Fertilization was ipsilateral to the side with the largest follicle as determined by ultrasound 12 hours after estrus; the side of ovulation was confirmed by detection of the corpus luteum by ultrasound 7-9 days after estrus. Pregnancy was determined by detection of the fetus by ultrasound after 42-45 days of estrus. This experiment was repeated 4 times and averaged over 3 fertilizer technicians. The side of ovulation was correctly determined in 205 out of 225 heifers (91%); surprisingly, the pregnancy rate was almost identical for ipsilateral and contralateral fertilization. Pregnancy rate is 1 × 10^Five2.5 × 10^Five, And 2.5 × 10⁶For sperm / fertilization (P> .1), they were 38/93 (41%), 45/87 (52%), and 25/45 (56%). There was a significant pregnancy rate difference (P <0.05) among technicians, but not between bulls. Sperm sorted by sex using a flow cytometer may be able to sufficiently reduce the number of sperm per fertilization using the described method with commercial application.
[0241]
As mentioned above and as can be seen from various experiments, the field is statistically justified, and thus various further experiments can be performed to show appropriate combination and restriction strategies. . Thus, synergies between various effects are further identified (eg, examples where the effects of dyes using laser excitation and the effects of combined dyes can be studied).
[0242]
The discussion contained in this application is intended to serve as a basic description. The reader should be aware that the specific discussion may not explicitly describe all possible embodiments; many alternatives are implicit. It may also not fully describe the general nature of the invention, and how each feature or element may actually exhibit a wider function, or a wide variety of alternatives or equivalents You do not have to explicitly show how the element can be shown. Again, these are implicitly included in this disclosure. Where the invention is described in device-oriented terms, each device element performs an implicit function. Apparatus claims may not only be included for the devices described, but method or process claims may also be included to handle the functions performed by the invention and each element. Neither the description nor the terminology is intended to limit the scope of the claims that may be filed. It should be understood that various changes can be made without departing from the essence of the invention. Such changes may also be implicitly included in the specification. These are still within the scope of the present invention. Extensive disclosure, including both the obvious embodiments shown, the vast variety of implicit alternative embodiments, the broad methods or processes, etc. is encompassed by the present disclosure.
[0243]
Moreover, each of the various elements of the invention and the claims can also be accomplished in various ways. It is understood that the present disclosure includes each such variation (variant embodiment of any instrument embodiment, method or process embodiment, or even a variation of any of these elements). Should. In particular, the disclosure referring to the elements of the present invention will understand that the words for each element may be expressed by equivalent instrument terms or method terms, even if only the function or result is the same. Should. Such equivalent, broader or even more general terms should be considered to be included in the description of each element or action. Such terms may be substituted where it is desired to clarify the implicit broad scope to which the present invention is entitled. It should be understood that all actions may be expressed as a means for obtaining the action or as an element that causes the action, rather than as an example. Similarly, each physical element disclosed should be understood to include a disclosure of the action that the physical element facilitates. It should be understood that rather than as an example of this aspect, the disclosure of “collector” includes disclosure of the action of “collecting”, whether or not explicitly discussed, and Conversely, where there is only a disclosure of the action of “collecting”, it should be understood that such disclosure includes disclosure of “collector”. It is understood that such changes and alternative terms are expressly included herein. Furthermore, in addition to the originally presented claims, it should be understood that the claims can be modified to at least more broadly address the following: i) the devices disclosed and described herein; ii) related methods disclosed and described, iii) and similar, equivalent, even implicit variations of each of these devices and methods, iv) achieving each of the functions indicated as disclosed and described These alternative designs, v) alternative designs and methods that achieve each of the functions shown as implicit to accomplish the disclosure and description, vi) shown as separate and independent inventions Each feature, element, and process, and vii) various combinations and permutations of each of the above.
[0244]
Various published citations may be helpful to help understand the present invention. These are listed below and are hereby incorporated by reference; however, these descriptions are expressly made by the applicant in that the descriptions may be considered inconsistent with this / patented patent of these inventions. It is not considered to be. US Pat. Nos. 5,660,997; 5,589,457; 5,514,537; 5,439,362; 5,346,990; 5,135,759; 5,212,244; 4,499,283; No. 4749458; No. 4698142; No. 4680258; No. 4511661; No. 4448767; No. 4362246; No. 4339434; No. 4276139; No. 4225405; No. 4155831; No. 4092229; No. 4085205; No. 4083957; No. 4067965; No. 40009260; No. 3889429; No. 3687806; Useful citations may also include the following publications:
[0245]
[Table 3]

Throughout this specification, unless the context otherwise requires, variations such as the word “comprise” or “comprises” or “comprising” refer to the element or integer, or group of elements or integers referred to. It is understood that it includes, but does not exclude any other element or integer, or group of elements or integers.
[0246]
【The invention's effect】
According to the present invention, sex-separated fertilization is achieved in a manner that operates at a lower dosage and in a realistic commercial environment.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of a sorter system based on a flow cytometer separation technique according to the present invention.
FIG. 2 is a diagram of cells entrained in the free fall region of a typical flow cytometer.
FIG. 3 is a conceptual diagram showing the difference that is almost shown as a result of the present invention.
FIG. 4 is a diagram of sorted cell flow as collected in the landing zone region.
[Explanation of symbols]
3 sheath liquid
14 Collection system
18 Sperm cells

Claims (32)

A method for sorting bovine or equine sperm cells using flow cytometry comprising the following steps:
a. Establishing a sperm cell source for supplying sperm cells to be sorted, the sperm cell source comprising a bovine or equine sperm cell source ;
b. Chemically tone made sheath fluid to produce a sheet over scan solution environment for the cells, and the cells comprising the steps of mixing with the sheath liquid, the sheath fluid is selected before and after sorting Prepared to be compatible with both cell fluid environments of
c. Detecting the characteristics of the cells in a flow cytometer;
d. Identifying sperm cells having desired characteristics at 5 00 Sort / sec to 1200 Sort / sec rate of; and e. Collecting sperm cells having the desired characteristics comprising the steps of :
Here, chemically preparing the sheath fluid to create a sheath fluid environment for the cells includes establishing a sheath fluid comprising 2.9% sodium citrate or HEPEs buffer. ,
The method wherein the desired feature is a sex feature . 2. The method of selecting sperm cells according to claim 1, wherein establishing the sperm cell source comprises establishing a source of non-repaired sperm cells. 3. The method of selecting sperm cells according to claim 2, wherein establishing the source of non-repaired sperm cells comprises establishing a source of sperm cells having non-transcribed DNA. 3. The method of selecting sperm cells according to claim 2, wherein establishing the source of non-repaired sperm cells comprises establishing a source of sperm cells having non-replicating DNA. 2. The method of sorting sperm cells according to claim 1, wherein establishing the sperm cell source comprises establishing a source of bovine sperm cells. 2. The method of sorting sperm cells according to claim 1, wherein the step of establishing a sperm cell source comprises establishing a source of equine sperm cells. The method of selecting a sperm cell according to any one of claims 1, 5 and 6, further comprising a step of fertilizing a non-human mammal using a dose of the sperm cell having the desired characteristics. Inclusion method. 8. The method of sorting sperm cells according to claim 7, wherein the fertilized sample comprises between 1 million and 3 million bovine sperm cells. 3. The method of sorting sperm cells according to claim 2, wherein the step of collecting cells having the desired characteristics comprises buffering the cells from impact using a collection container. 10. A method for sorting sperm cells according to claim 9, wherein buffering the cells from impact using the collection container comprises providing a wide opening in the container. 6. A method for sorting sperm cells according to claim 5, further comprising the step of using collected cells having the desired characteristics for artificial insemination in non-human mammals. 7. A method for sorting sperm cells according to claim 6, further comprising using the collected cells having the desired characteristics for artificial insemination in a non-human mammal. The method of sorting sperm cells according to claim 1, wherein the step of minimizing chemical changes provided as a result of the cells being subjected to the sheath fluid is particularly reactive. comprises at least one over-reactive chemical composition, comprising the step of providing a cell fluid environment after sorting before and sorting, wherein the step of manufactured chemically regulating the sheath liquid, the A method of minimizing changes to overreactive chemical compositions. A method for sorting sperm cells according to any one of claim 1 to 5, wherein the sheet over scan solution chemically step of steel tone, 2.9% of sodium citrate Establishing a sheath fluid comprising: 15. A method for sorting sperm cells according to claim 14, wherein the step of establishing a cell source supplying the sorted cells is overreactive to a chemical composition in the surrounding fluid environment. Providing a cell source, the method comprising establishing a cell source. A method for sorting sperm cells according to any one of claims 1 or 6, wherein the sheet over scan solution chemically step of steel tone, establishing a sheath fluid containing HEPEs buffered medium Including the method. 17. A method for sorting sperm cells according to claim 16, wherein the step of establishing a cell source supplying the sorted cells is overreactive with chemical compositions in the surrounding fluid environment. Providing cells, establishing a cell source. A method for sorting sperm cells according to claim 1, wherein 5 00 sort / sec to 1200 Sort / sec step of selecting the cells at a rate of at least 50 pounds per square inch the cells (344 The method further comprises subjecting to a pressure of .7 kilopascal). A method for sorting sperm cells according to claim 1, wherein establishing the fine胞供sources comprises the selection before the fluid environment, the step of collecting cells having the desired characteristics is the A method comprising a fluid environment after sorting. A method for sorting bovine or equine sperm cells by flow cytometry comprising the following steps:
a. Establishing a cell source supplying cells to be sorted, the sperm cell source comprising a bovine or equine sperm cell source ;
b. Establishing a sheath fluid for creating a sheath fluid environment for the cells and mixing the sheath fluid with the cells;
c. Detecting the characteristics of the cells in a flow cytometer;
d. Identifying a cell having the desired characteristics at 5 00 speed sorting / sec to 1200 Sort / sec;
e. Collecting cells having the desired characteristics in a collection fluid; and f. The the collection fluid chemically regulated manufactured to be compatible with respect to the sheath fluid, compatible with selected previous fluid environment, the step of producing a final collection fluid environment for said cells ,
A method, including,
Wherein establishing a sheath fluid to create a sheath fluid environment for the cells includes establishing a sheath fluid comprising 2.9% sodium citrate or HEPEs buffer;
Wherein said desired Ru sexual characteristics der method. 21. The method of sorting sperm cells of claim 20, wherein establishing the cell source includes providing initial nutrients to the cells, and further providing collected fluid nutrients to the cells. Wherein collecting the cells having the desired characteristics in the collection fluid averages the initial nutrient and the collected fluid nutrient after completion of the step of collecting the cells. A method comprising the steps of: 22. The method of sorting sperm cells according to claim 21, wherein the step of collecting cells having the desired characteristics in the collection fluid comprises 6% egg yolk prior to initiating the collection step. Establishing a citrate collection fluid. 23. A method for sorting sperm cells according to claim 22, wherein the step of establishing a cell source includes the step of establishing a source of bovine sperm cells. A method for sorting sperm cells according to claim 23, further comprising the step of fertilizing a mammal using the sperm cells a dose having the desired characteristics, methods. 24. A method for sorting sperm cells according to claim 23, wherein said step of establishing sheath fluid to create a sheath fluid environment for said cells comprises 2.9% sodium citrate. A method comprising establishing a liquid. 21. A method for sorting sperm cells according to claim 20, wherein the step of establishing a cell source supplying the sorted cells is overreactive to a chemical composition in the surrounding fluid environment. Providing cells, establishing a cell source. 21. A method for sorting sperm cells according to claim 20, wherein the step of collecting cells having the desired characteristics further comprises buffering the cells from impact using a collection container. 28. A method of sorting sperm cells according to any one of claims 9 or 27, wherein the step of buffering the cells from impact using the collection container avoids impact between the cells and the collector. A process comprising sizing the collector. 28. A method of sorting sperm cells according to any one of claims 9 or 27, wherein the step of buffering the cells from impact using the collection container comprises providing a wide collection tube. ,Method. 30. A method of sorting sperm cells according to claim 29, wherein providing the wide collection tube comprises providing a collection tube having a diameter of at least 15 mm. 28. A method of sorting sperm cells according to claim 27, wherein said step of buffering said cells from impact using said collection container comprises providing a container adapted for flow. 21. A method for sorting sperm cells according to claim 20, wherein the step of identifying cells having the desired characteristics comprises identifying sperm cells stained using a tens of micromolar amount of dye. Including the method.

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